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Chiara Gualandi

Porous Polymeric
Bioresorbable Scaffolds for
Tissue Engineering

Doctoral Thesis accepted by

University of Bologna, Italy

123
Author Supervisor
Dr. Chiara Gualandi Prof. Mariastella Scandola
Department of Chemistry Department of Chemistry
University of Bologna University of Bologna
Via Selmi 2 Via Selmi 2
40126 Bologna 40126 Bologna
Italy Italy
e-mail: c.gualandi@unibo.it e-mail: mariastella.scandola@unibo.it

ISSN 2190-5053 e-ISSN 2190-5061

ISBN 978-3-642-19271-5 e-ISBN 978-3-642-19272-2

DOI 10.1007/978-3-642-19272-2

Springer Heidelberg Dordrecht London New York

 Springer-Verlag Berlin Heidelberg 2011

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Parts of this thesis have been published in the following journal articles

Gualandi C., Wilczek P., Focarete M.L., Pasquinelli G., Kawalec M., Scandola M.
‘‘Bioresorbable electrospun nanofibrous scaffolds loaded with bioactive mole-
cules’’ e-Polymer, publication no.: 060[2009] (Reproduced with Permission).
Zucchelli A., Fabiani D., Gualandi C., Focarete M.L. ‘‘An innovative and versatile
approach to design highly porous, micropatterned, nanofibrous polymeric mate-
rials’’ Journal of Materials Science 2009, 44, 4969-4975 (Reproduced with
Permission).
Focarete M.L., Gualandi C., Moroni L. ‘‘Working with electrospun scaffolds: some
practical hints for tissue engineers’’ chapter 2, in ‘‘Electrospun Nanofibers
Research: Recent Developments’’ A.K. Haghi Editor, Nova Science Publishers
(Hauppauge, NY) (2009), p. 19-34 (Reproduced with Permission).
Gualandi C., White L. J., Chen L., Gross R. A., Shakesheff K. M., Howdle S. M.,
Scandola M. ‘‘Scaffold for tissue engineering from non-isothermal supercritical
carbon dioxide foaming of a highly crystalline polyester’’ Acta Biomaterialia
2010, 6, 130-136 (Reproduced with Permission).
Foroni L., Dirani G., Gualandi C., Focarete M.L., Pasquinelli G. ‘‘Paraffin
embedding allows effective analysis of proliferation, survival and immunophe-
notyping of cells cultured on poly(L-lactic acid) electrospun nanofiber scaffolds’’
Tissue Engineering part C: Methods 2010, 16(4), 751-760 (Reproduced with
Permission).
Focarete M.L., Gualandi C., Scandola M., Govoni M., Giordano E., Foroni L.,
Valente S., Pasquinelli G., Gao W., Gross R.A. ‘‘Electrospun scaffolds of a
polyhydroxyalkanoate consisting of x-hydroxylpentadecanoate repeat units:
fabrication and in vitro biocompatibility studies’’ Journal of Biomaterials Science,
Polymer Edition 2010, 21, 1283-1296 (Reproduced with Permission).
Supervisor’s Foreword

Tissue engineering, and regenerative medicine in general, are new challenges of

modern medical science which aims to provide solutions to human society clinical
needs. Since the beginning of these fields three decades ago, a huge amount of
funding has been distributed worldwide to the scientific community with the final
goal of creating an organ or part of it in vitro, or of inducing in vivo regeneration.
Some of these research efforts have been awarded by seeing in vitro engineered
tissues becoming commercial products that nowadays help people all around the
world in completely or partially restoring the function of biological tissues.
Despite the continuous expansion of tissue engineering (attested by an impres-
sively growing number of publications and by the many academic institutions and
industries working in the field), on the whole this area has been progressing
slowly, probably due to the extremely difficult problems raised by the challenge of
regenerating a biological tissue. Therefore, even if tissue engineering cannot be
considered as a ‘‘new’’ discipline, the field needs innovative concepts and original
approaches to overcome the present limits and face new challenges.
In the tissue engineering approach, biomaterials and scaffolds obtained there-
fore play a central role, i.e. they create the biophysical and biochemical envi-
ronment suitable to direct cellular behavior and function. Success of an engineered
tissue construct strongly depends on scaffold features and this is the reason why
the main aim of this Ph.D. thesis was to develop innovative bioactive nano-
structured constructs. The research activities, carried out not only at the Chemistry
Department ‘‘G. Ciamician’’ (University of Bologna) but also at the Inorganic and
Materials Chemistry Department and Centre of Biomolecular Science (University
of Nottingham) and at the Polymers and Biomaterials Lab (University of Man-
chester), dealt with the manipulation of polymer chemical structure in order to tune
material physical properties, and with optimization of scaffold 3D architecture by a
smart use of different scaffold fabrication techniques. This thesis illustrates how a
rational use of two alternative technologies, i.e. supercritical carbon dioxide
foaming and electrospinning, can lead to fabrication of innovative polymeric
bioresorbable scaffolds made of commercial or ‘ad-hoc-synthesized’ hydrolysable

vii
viii Supervisor’s Foreword

polyesters. The thesis also explores the possibility of making these new scaffolds
bioactive by suitable functionalization procedures.
The multidisciplinary nature of this research is imperative in pursuing the
challenge of tissue regeneration successfully. One of the strengths of this thesis is
the integration of knowledge from chemistry, physics, engineering, materials
science and biomedical science which has contributed to setting up new national
and international collaborations, while strengthening existing ones.

Bologna, March 2011 Mariastella Scandola

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Tissue Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Biological Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 The Scaffold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3.1 Biomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.3.2 Scaffold Fabrication Technologies. . . . . . . . . . . . . . . . . 13
1.4 Scaffold Functionalization . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2.2 Scaffold Fabrication by ScCO2 Foaming . . . . . . . . . . . . . . . . . 33
2.3 Scaffold Fabrication by Electrospinning . . . . . . . . . . . . . . . . . . 35
2.3.1 Surface Modification . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.3.2 In Vitro Degradation Experiments. . . . . . . . . . . . . . . . . 37
2.3.3 Scaffold Preparation for Cell Culture Experiments . . . . . 37
2.4 Characterization Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2.4.1 Thermogravimetric Analysis. . . . . . . . . . . . . . . . . . . . . 39
2.4.2 Differential Scanning Calorimetry . . . . . . . . . . . . . . . . . 39
2.4.3 Scanning Electron Microscopy . . . . . . . . . . . . . . . . . . . 39
2.4.4 Micro X-Ray Computed Tomography . . . . . . . . . . . . . . 40
2.4.5 Stress–Strain Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 40
2.4.6 Wide Angle X-Ray Diffraction . . . . . . . . . . . . . . . . . . . 40
2.4.7 Gel Permeation Chromatography . . . . . . . . . . . . . . . . . 40
2.4.8 f-Potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . ....... 43

3.1 Porous Scaffold Fabrication by ScCO2 Foaming . . . . . ....... 44
3.2 Porous Scaffold Fabrication by Electrospinning. . . . . . ....... 54
3.2.1 Process Conditions Optimization: The Example
of P(PDL-CL). . . . . . . . . . . . . . . . . . . . . . . . ....... 57

ix
x Contents

3.2.2 Electrospun Polymers . . . . . . . . . . . . . . . . . . . . . . . . . 62

3.2.3 Fibre Morphologies . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
3.2.4 Fibre Deposition Geometries . . . . . . . . . . . . . . . . . . . . 72
3.2.5 ‘‘Composite’’ Electrospun Scaffolds . . . . . . . . . . . . . . . 78
3.2.6 Electrospun Scaffold Functionalization . . . . . . . . . . . . . 81
3.3 Hydrolytic Degradation Experiments . . . . . . . . . . . . . . . . . . . . 87
3.4 Cell Culture Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
3.4.1 Scaffold Preparation for Cell Culture Experiments . . . . . 94
3.4.2 Characterization of Cells Cultured on Electrospun
Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 98
3.4.3 Biocompatibility Evaluation of Electrospun Scaffolds:
The Example of PPDL . . . . . . . . . . . . . . . . . . . . . . .. 100
3.4.4 Electrospun Fibres Loaded with Growth Factor: Effect
on Stem Cell Culture. . . . . . . . . . . . . . . . . . . . . . . . .. 103
3.4.5 Effect of Electrospun Fibre Orientation on Cancer
Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 107
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 111

4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
List of Abbreviations

ATRP Atom transfer radical polymerization

CE 2-Chloroethanol
CLF Chloroform
Cp Heat capacity
d Needle to collector distance
DCM Dichloromethane
DMEM Dulbecco’s modified eagle’s medium
DMF N,N-dimethylformamide
DMSO Dimethyl sulfoxide
DSC Differential scanning calorimetry
ECGS Endothelial cell growth factor supplement
ECM Extracellular matrix
ES Electrospinning/electrospun
EtOH Ethanol
FFPE Formalin fixed paraffin embedding
GF Growth factor
GMMA Glycerol monomethacrylate
GPC Gel permeation chromatography
HA Hydroxyapatite
Hc Crystallization enthalpy
HFIP 1,1,1,3,3,3-Hexafluoro-2-propanol
Hm Melting enthalpy
l-CT Micro X-ray computed tomography
MetOH Methanol
MI ATRP Macroinitiator
Mn Number average molecular weight
Mw Weight average molecular weight
P(D,L)LA Poly(D,L)lactide
P(L)LA Poly(L)lactide
P(LA-TMC) Poly((L)lactide-co-trimethylencarbonate)
P(PDL-CL) Poly(x-pentadecalactone-co-e-caprolactone)

xi
xii List of Abbreviations

P(PDL-DO) Poly(x-pentadecalactone-co-p-dioxanone)
PBS Phosphate buffered saline
PCL Poly(e-caprolactone)
PCR Polymerase chain reduction
PDI Polydispersity index
PEO Poly(ethylene oxide)
PGA Polyglycolide
PLA Polylactide
PLA-T6 Six-arms star-branched oligo(D,L)lactic acid
PLAxCLy Poly(lactide-co-e-caprolactone) copolymers, where x and y
indicate mol% content of the comonomers
PLAxGAy Poly(lactide-co-glycolide) copolymers, where x and y
indicate mol% content of the comonomers
PPDL Poly(x-pentadecalactone)
PTMC Poly(1,3-trimethylene carbonate)
R Solution flow rate
RH Relative humidity
RT Room temperature
ScCO2 Supercritical carbon dioxide
SEM Scanning electron microscopy
SI-ATRP Surface initiated atom transfer radical polymerization
Tc Crystallization temperature
TCP b-Tricalcuim phosphate
TCPS Tissue culture polystyrene
TE Tissue engineering
Tg Glass transition temperature
TGA Thermogravimetric analysis
THF Tetrahydrofuran
Tm Melting temperature
WAXD Wide angle X-ray diffraction
DV Applied voltage
Chapter 1
Introduction

1.1 Tissue Engineering

Tissue or organ failure is one of the most frequent and devastating problems in
medicine. Current therapeutic approaches (allografts, xenografts, autografts and
implantation of biomedical devices) have largely improved the quality of life but
they are associated with clear limitations including donor availability, infection,
poor integration and potential rejection of the implant. Regenerative medicine was
developed with the aim to overcome such limitations and to find revolutionary and
powerful therapies for the treatment of tissue diseases, with the basic idea to repair
or re-create tissues or organs in order to restore impaired functions [1, 2].
Since its origin, regenerative medicine has rapidly grown and has attracted the
interest of many scientists and surgeons throughout the world. Nowadays regen-
erative medicine encompasses different strategies for the creation of new tissue
including the use of cloning, of isolated cells, of non-cellular structures and of
cells constructs. The latter approach, which is usually referred to as tissue
engineering (TE), is believed to be highly promising for regenerating tissues. It is
pointed out that a clear distinction between TE and regenerative medicine does not
exist in the literature and some scientists use these terms as synonyms. In the
present project, however, TE will be considered a sub-discipline of regenerative
medicine that intends to use cell-constructs to achieve tissue repair.
Even if the term ‘‘tissue engineering’’ was firstly coined in the mid of 1980s, it
became part of the scientists common language only in 1993, when Langer and
Vacanti [3] defined TE as ‘‘an interdisciplinary field that applies the principles of
engineering and life sciences toward the development of biological substitutes that
restore, maintain, or improve tissue function or a whole organ’’. Figure 1.1 sket-
ches TE approach for the preparation of cell constructs: cells are cultured in vitro
under precisely controlled culture conditions on a porous three-dimensional (3D)
material that acts as scaffold for cell growth and proliferation. Once being

C. Gualandi, Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering, 1

Springer Theses, DOI: 10.1007/978-3-642-19272-2_1,
 Springer-Verlag Berlin Heidelberg 2011
2 1 Introduction

Fig. 1.1 TE approach by using cell-scaffold construct

implanted in the human body, the scaffold is eventually bioresorbed and the space
occupied by it is replaced by new tissue produced by cells [4, 5].
Significant progress has been realized in TE since its principles were defined
and to date several products, incorporating cells together with scaffold, have
gained regulatory approval. Commercial devices are mostly dedicated to skin,
bone and cartilage engineering whereas products for cardiac, nerve, kidney or
pancreas engineering are not widespread yet [6].
The obtainment of an engineered tissue encompasses the optimization of a huge
number of factors and parameters that interplay in affecting the success or the
failure of the construct. Since an engineered tissue is basically composed of cells
seeded on a scaffold, the design of a cell-scaffold construct firstly requires
the selection of both suitable cell type and porous scaffold, depending on the
characteristics of the tissue that has to be replaced.
In terms of cell origin, cells can be autologous (patient’s own cells harvested by
biopsy), allogeneic (cells from other human sources) or xenogeneic (cells from
different species), although the last two, being genetically different from patient’s
cells, may induce immunorejective response. Moreover, cells exist in a differen-
tiate state or they can be undifferentiated (stem cells). In the first case cell
phenotype and functions are well defined but this is accompanied with a limited
capability to proliferate. On the contrary stem cells can self-renew for long periods
of time and can be induced to differentiate upon exposure to specific cues [7].
The rapid expansion of interest in stem cells arises from their proliferative and
developmental potential that promises an essentially unlimited supply of specific
cell types for both basic research and transplantation therapies.
The second basic component of an engineered tissue is the scaffold that acts as a
temporary template, guiding cell organization, growth and differentiation and
providing structural stability and a 3D environment where cells can produce new
biological tissue. Scaffolds may be created from various types of materials,
1.1 Tissue Engineering 3

including natural and synthetic polymers and inorganic substances. The fabrication
technique controls the morphology of the porous scaffold that can be made of
fibres, gels or can be obtained by creating pores within a polymer matrix.
Additional components of cell-scaffold constructs can be biomolecules that are
introduced either as scaffold additives to be delivered during cell culture or by
functionalizing scaffold surface. Biomolecules (e.g. growth factors, peptide
sequences, gene vectors, etc.) are used in order to induce specific cell behaviour
(e.g. enhancing cell adhesion, cell proliferation, cell differentiation, etc.).
Another important aspect in the fabrication of an engineered tissue are cell
culture conditions that are known to influence cell growth. In particular, dynamic
cell culture conditions improve the development of a more tissue-like construct
with respect to static conditions, thanks to efficient nutrition and exogenous stimuli
that direct cellular activity and phenotype [8–10]. Dynamic cell cultures are
performed with bioreactors, where culture conditions, such as mass transport
throughout the scaffold, mechanical stimulation, electrical stimulation etc., can be
precisely controlled.
In summary, several critical elements should be considered in order to achieve
successful regeneration of damaged tissue, including cell type and source, scaffold
material and 3D structure, biomolecules and cell culture conditions (Fig. 1.2).
Hence, it is clear that TE studies require a multidisciplinary approach that must
involve not only biological and medical expertises but also competences in
engineering, chemistry and materials science.
The present research project focuses its attention on the fabrication of polymeric
bioresorbable scaffolds by means of different techniques, with a particular con-
sideration towards electrospinning (ES) technology. Knowledge of polymer science
has been applied to produce, manipulate and characterize polymeric scaffolds.
Given the multidisciplinary character of this field, the research was carried out with
the cooperation of experts in other disciplines other than polymer science, such as
mechanical and electrical engineering and biochemistry. In particular, engineers
contributed to improve scaffold fabrication technology whereas biochemists

Fig. 1.2 TE key elements

4 1 Introduction

performed in vitro biological experiments making use of the polymeric scaffolds

produced in the context of this project.

1.2 Biological Tissues

A detailed understanding of the structure and function of normal biological tissues is

central to the design of artificial organs and to the development of tissue engineering
strategies. In general, biological tissues are composed of cells and of a non-cellular
part called extracellular matrix (ECM). Physiological functions in the human body
are coordinated by different types of organs composed of a great variety of mam-
malian cells surrounded by ECM, which displays different chemical compositions
and different spatially organized configurations depending on the type of organ.
Cells are tissue building blocks and a considerable part of TE studies deal with
their isolation from native tissues and their expansion in vitro. Since most cells do
not grow efficiently in suspension but they need to be attached to a substratum
[11], the common procedure is to seed cells on artificial bidimensional substrates
called tissue culture polystyrene (TCPS) plates. Once enough cells are obtained,
they are harvested from the 2D plates and then seeded on 3D scaffolds.
In conventional TE approaches fully differentiated adult cells, which are estimated
to be more than 210 types in mammalian body [7], are employed. However, given
the shortage of adult human cells due, in particular, to difficult harvesting pro-
cedure and their limited survival capability in vitro, modern TE approaches
involve the use of stem cells, i.e. of undifferentiated cells that easily duplicate in
vitro and that can be induced to specialize following exposure to specific cues [2].
Cell behaviour is deeply influenced by hierarchical patterns and by physico-
chemical properties of the environment (Fig. 1.3). In all biological tissues, cells
are surrounded by ECM that is composed of carbohydrates and proteins locally
secreted by cells and assembled in an organized network. For the sake of
simplicity ECM can be considered composed of (1) a gel-like component, (2) a
fibrous component and (3) specialized proteins [12]. The highly viscous and
hydrated gel-like part confers lubricant and hit-absorption properties to the ECM.
It consists of carbohydrates assembled to form polysaccharides commonly called
glycosaminoglycans which, in turn, are covalently attached to a protein backbone
to form proteoglycans. The fibrous component of ECM is essentially composed of
collagen and elastin that create a complex network of fibres with diameters ranging
from few to hundreds of nanometers, imparting rigidity and strength to the entire
tissue. Finally, ECM holds proteins such as growth factors (GFs), cytokines,
enzymes and multidomain proteins (e.g. fibronectin, laminin, vitronectin, etc.) that
play a key function in the communication with the surrounding cells.
Besides providing structural support to cells, ECM plays a central role in
modulating cell behaviour and in maintaining tissue architecture and functions
thanks to its dynamic interaction with cells. Indeed, cells continuously interact
with the external environment via membrane proteins (receptors, e.g. integrins)
1.2 Biological Tissues 5

Fig. 1.3 Schematic representation of the reciprocal molecular interaction between cell and its
surrounding (reprinted from [12], Copyright (2005), with permission from Macmillan Publishers
Ltd: Nature Biotechnology)

that bind external proteins (ligands, e.g. fibronectin) located both on the surface of
surrounding cells and in the ECM, by following a lock-and-key mechanism.
Through an intracellular cascade of reactions, the ligand-receptor interaction is
translated into a specific signal to guide the cell to a specific activity [13]. This
dynamic interaction allows to finely control cell fate, shape and behaviour in
response to even small changes in ECM composition [14–17]. Overall, ECM
functions can be summarized as follows [18]:
• establishment of a hierarchical patterned micro/nanoenvironment;
• mechanical and structural support;
• regulation of cell shape and cell polarity;
• storage of regulatory molecules (enzymes, GFs, multidomain proteins);
• regulation of cell function (e.g. proliferation, growth, survival, migration, and
differentiation).
Given the central role of ECM and of cell environment in determining cell
response and behaviour, it is quite evident that biologists and biochemists need to
deeply understand the biological phenomena that rule cell-ECM and cell–cell
interactions. It is also clear that cells must be provided with a scaffold, having suitable
6 1 Introduction

biological and mechanical features to ensure cell attachment, proliferation and

spontaneous deposition of ECM by cells. To this aim, materials science plays its role
in TE field, by fabricating an ECM-substitute scaffold with appropriate physical and
chemical properties and with a proper 3D structure and architecture.

1.3 The Scaffold

The officially accepted definition considers a scaffold as a ‘‘support, delivery

vehicle or matrix for facilitating the migration, binding or transport of cells or
bioactive molecules used to replace, repair or regenerate tissues’’ [19]. Indeed,
recent scaffolds are not intended only to support cell growth but they can also load
bioactive molecules having a specific biological function. This aspect will be
deeply discussed in Sect. 1.4. Moreover, it is worth noting that last generation
scaffolds are intended to be as much biomimetic as possible, in terms of
(1) mechanical performances, (2) 3D morphology and (3) surface chemistry, and
they are designed according to this scope.
It is well-accepted that an ideal functional scaffold should meet the following
challenging requirements [20–23]:
• to be biocompatible;
• to have mechanical properties consistent with those of the tissue it replaces;
• to be bioresorbable;
• to degrade at a rate matching that of new tissue formation;
• to have proper surface properties to enable cell attachment, growth, prolifera-
tion, and differentiation as well as to promote extracellular matrix formation;
• to have the optimum architectural properties in terms of pore size, porosity, pore
interconnectivity, and permeability in order to allow efficiently delivery of
nutrients and removal of waste.
The imperative requirement for a scaffold is to be biocompatible. Since the
scaffold works in contact with leaving cells in vitro and with tissue, once
implanted in vivo, it must not elicit harmful response from the biological envi-
ronment, i.e. it should interact with cells and host tissue without inducing cyto-
toxicity or adverse immune response.
Scaffold mechanical properties are another key element that distinguishes a
successful implant from a failed one. Indeed, since many tissues undergo
mechanical stresses and strains, it is extremely important that mechanical prop-
erties of the scaffold match as closely as possible those of the tissue intended to
regenerate, so that formation of new ECM is not limited by mechanical failure of
the scaffold. Moreover, a good mechanical transfer between the scaffold and the
new forming tissue is required in order to provide sufficient mechanical stimula-
tion for ensuring a proper tissue growth [24].
Scaffold is intended to be a temporary support that is eventually replaced in
the organism by new regenerated tissue. Hence, scaffold must be degradable in the
1.3 The Scaffold 7

human body through molecular fragmentation mechanisms that results in

the formation of degradation by-products and the gradual disappearance of the
scaffold. Degradation can occur via hydrolysis or it can be mediated by enzymes,
depending on polymer chemical structure. In any case, the long-term success of an
engineered tissue will be achieved only if degradation products are completely
resorbed by the organism by naturally occurring metabolic pathways, i.e. if the
scaffold is bioresorbable [25]. Moreover, scaffold degradation rate should mirror
the rate of tissue formation. This criterion is extremely difficult to achieve but it is
particularly important as regards the structural supporting role of the scaffold.
Indeed, in order not to compromise the integrity of the implant, the scaffold should
maintain its structural function until the regenerated tissue can assume its
supporting role and, over time, the degrading scaffold should gradually transfer its
function of load bearing to the new forming tissue. The control of scaffold
mechanical properties over time and during degradation process remains one of the
greatest challenges in tissue engineering [26, 27].
Another significant feature of the scaffold is its surface properties which are
directly connected to its capability of creating a chemically suitable environment
that promotes cell adhesion, migration and proliferation in order to obtain an
entirely colonized 3D cell construct. Cell adhesion is always a receptor-mediated
process that occurs via interaction between membrane proteins, called integrins,
and multidomain proteins that act as ligands. In natural tissues typical ligands
dislocated in the ECM are fibronectin, laminin and vitronectin that bind integrins,
forcing the cells to attach to the ECM [28, 29]. Cell-scaffold interaction similarly
occurs. Indeed, when a scaffold gets in contact with a biological environment the
first occurring event is protein absorption as a result of Van der Waals, hydro-
phobic and electrostatic interactions, and hydrogen bonding [30]. These interac-
tions depend on scaffold surface, namely on its chemical composition, roughness
and topography [31]. Therefore cell-scaffold interactions are related to the com-
position of the protein layer attached to the scaffold surface. It has been largely
demonstrated that polymers can adsorb many proteins [32] and it is also possible to
adapt a material presenting good bulk properties by improving its surface prop-
erties towards cell adhesion through surface modification. Many techniques have
been developed to modify material surfaces such as plasma or ion treatment but,
more recently, ECM biomolecules, such as proteins, peptides or growth factors,
have been immobilized on scaffold surface with the aim to obtain bioactive and
biomimetic scaffolds [33]. Surface modification approaches will be more exten-
sively discussed in Sect. 1.4.
Cell colonization of the scaffold depends not only on scaffold surface properties
but also, indirectly, on its 3D architecture. Porosity (the amount of void space),
size, geometry, orientation and interconnectivity of pores and channels directly
affect the transport and delivery of nutrients for cells throughout the scaffold [34].
In particular high porosity, high surface area to volume ratio and high pore
interconnectivity are required in order to ensure uniform tissue ingrowth, efficient
delivery of nutrients to the interior of the scaffold and removal of waste towards
the exterior [35]. The supply of nutrients and oxygen is realized in vivo by the
8 1 Introduction

blood vascular system. The achievement of vascularization of 3D scaffolds is still

one of the greatest challenges in TE.
The above described scaffold characteristics (i.e. biocompatibility, mechanical
properties, bioresorbability, surface properties and architecture) are strictly related
to two major factors that interplay in controlling scaffold properties: (1) the type of
polymer material and (2) the scaffold fabrication technology. For instance, toxic
residual substances can be released out from the scaffold causing harmful effects.
Such substances can be either monomers or impurities in the starting material or
substances deriving from material processing (e.g. degradation products, organic
solvents, etc.) Mechanical properties primary depend on the raw material but they
can dramatically change according to scaffold architecture which, in turn, is
determined by the technique employed to produce the scaffold. Another example is
bioresorbability and, in particular, the degradation rate that depends not only on
polymer properties (i.e. chemical composition, monomer distribution and micro-
structure in copolymers and molecular weight) but also on scaffold architecture
(i.e. scaffold dimension and pore walls).
It is clear that the correct design of a scaffold for a specific application requires
to accurately know which properties it should exhibit in order to successfully
achieve its function. Once the necessary scaffold features are clearly defined,
material science intervenes in selecting the proper polymer material, the suitable
scaffold fabrication technology and the appropriate treatments in order to obtain a
scaffold that matches as closely as possible the specific requirements.
The following subchapters outline the principles of biomaterials science and
describe the more frequently used biomaterials for tissue engineering applications,
with particular attention towards polymeric ones. Common scaffold fabrication
approaches are introduced, focusing mainly on electrospinning and supercritical
carbon dioxide technology.

1.3.1 Biomaterials

A biomaterial can be considered a synthetic material used to make a device

designed to replace a part or a function of the body. The commonly accepted
definition of biomaterial was proposed at the Conference of the European Society
for Biomaterials (England, 1986): ‘‘any substance, other than a drug, or combi-
nation of substances, synthetic or natural in origin, which can be used for any
period of time, as a whole or as a part of a system which treats, augments, or
replaces any tissue, organ, or function of the body’’ [36].
The basic requirement of a biomaterial is to be biocompatible, namely ‘‘to
perform with an appropriate host response in a specific application’’ [36]. The
biomaterial itself, but also additives or degradation products, must not cause
harmful reactions in contact with the body. For this reason the design and the
fabrication of a biomedical device, in terms of synthesis of the raw materials but
also of manufacture technologies, must respect, first of all, the biocompatibility
1.3 The Scaffold 9

requirement. Biocompatibility must be tested and the devise must be approved by

appropriate regulatory agencies (such as the Food and Drug Administration, USA)
before it can be marketed.
According to the definition of biomaterial reported above, the following devices
fall within this category:
• implantable devices, e.g. dental implants, pacemakers, orthopedic and vascular
prostheses, etc.;
• devices working in contact with biological tissues/fluids for a limited period of
time, e.g. surgical instruments, catheters, contact lenses, sutures, etc.;
• devices for extra-body treatments, e.g. dialysis membranes, blood vessels.
According to their chemical nature, synthetic biomaterials can be classified as:
(1) polymers, (2) metals, (3) ceramics and (4) composites. Table 1.1 reports some
examples of applications, together with some considerations about advantages and
disadvantages related to each class of biomaterial.
Thanks to the continuous progress in molecular biology and in the compre-
hension of cell-biomaterial interactions, since its birth, biomaterial science has
seen an extraordinary evolution in the development of increasingly biocompatible,
bioactive and specifically functional biomedical devices. The improvement of
biomaterial features went through three different stages, each concerning different
purposes [38]. The ‘‘first generation’’ of biomaterials, developed in 1960s as
implantable devices, were designed to possess suitable physical properties in view
of their function as organ substitutes, and they were intended to be inert, namely to

Table 1.1 Categories of biomaterials (adapted from [37])

Materials Uses Advantages Disadvantages
Polymers
Polyammides, silicon Sutures, blood vessels, Resilient Not strong
rubber, polyesters, catheters, devices for drug Easy to fabricate Deformation
polyurethans, delivery, contact lens, etc. Wide range of with time
polytetrafluoroethylene, mechanical
polymethylmetharylate, properties
etc. Biodegradable
Metals
Ti and its alloys, stainless Joint replacements, bone Strong May corrode
steels, Au, Ag, Co–Cr plates and screws, dental Tough High densities
alloys, etc. implants, surgical Ductile
instruments, etc.
Ceramics
Aluminium oxides, calcium Dental implants, femoral Strong under Brittle
phosphates, heads, coating of compression Not resilient
hydroxyapatite, orthopaedic devices Good trybological
Bioglasses, etc. properties
Composites
Joint implants, tendon and hip Strong Difficult to
replacements, heart valves Tailor-made fabricate
10 1 Introduction

cause minimal tissue reactions. Later in the 1980s, the ‘‘second generation’’ of
biomaterials was born with the necessity not only to be tolerated by the organism,
but also to elicit desired and controlled response by the biological tissue, again
with no harmful effects. The so called bioactive materials were used, for example,
in orthopaedic surgery (Bioglass). Moreover, in this period problems connected
with long term tissue-biomaterial interactions were minimized with the develop-
ment of bioresorbable materials that were used also as drug delivery systems.
Subsequently biomaterials have evolved into their ‘‘third generation’’ and
nowadays they are intended to stimulate highly precise reactions at the molecular
level with biological tissues. With this aim it is clear that precise surface engi-
neering and nanotechnology are needed in order to build tailored architectures for
specific applications. Scaffolds for tissue engineering can be considered as ‘‘third
generation’’ biomaterials.
For this reason it is clear that raw biomaterials suitable to produce bioresorbable
scaffolds are exclusively either biodegradable polymers (either natural or synthetic
ones) or biodegradable ceramics (calcium phosphates) that must disappear in the
human body as a consequence of hydrolytic and/or enzymatic degradation,
whereas metals and all the other non-degradable materials are not used in this
context.
Biodegradable ceramics, most of all hydroxyapatite (HA) and b-tricalcium
phosphate (b-TCP), are naturally found in bone and they are used in combination
with degradable polymers to make composite scaffolds for bone tissue regenera-
tion [39–41]. Indeed, these inorganic substances are often difficult to be processed
by their own into highly porous structures. Moreover, their inherent brittleness
limits their use as plain materials for scaffold fabrication, so that, in order to obtain
structural resistant supports, it is necessary to combine them with polymer
matrices [21, 42].
Natural polymers such as proteins (e.g. collagen, gelatin, fibrin, starch, etc.),
polysaccharides (e.g. hyaluronic acid, alginate, chitin, etc.) and bacterial polyesters
(e.g. polyhydroxybutyrate, polyhydroxyvalerate, etc.) are widely used to produce
scaffolds with different topography, either as pure materials or in combination with
synthetic polymers or inorganic substances [43–47]. The use of natural polymers
ensures excellent bioactivity towards biological environment because of their
inherent properties of biological recognition. In particular, if ECM polymers, such
as collagen or hyaluronic acid, are employed, scaffolds that mimic the chemical
properties of natural ECM are obtained (biomimicry). Biomimetic features are so
relevant that, besides pure natural polymers, decellularized ECM, containing
multiple natural macromolecules, is also used as scaffold for tissue repair. Despite
the incredible advantage of biomimicry, some issues, associated with purification,
pathogen transmission, processability into porous structure and sustainable pro-
duction, restrict the use of natural polymers for scaffold fabrication. Moreover,
poor control of mechanical properties and degradation rate limits the possibility to
tailor their employment to specific functions.
Some problems associated with the use of natural materials can be overcome
with the employment of synthetic polymers. Indeed, the latter, besides being less
1.3 The Scaffold 11

expensive and better processable, can be synthesized ad hoc with a precise control
of molecular structure to tune mechanical and degradation properties. Further-
more, polymers can be synthesized with specific functional groups in order to
make them bioactive towards biological environment. These characteristics make
synthetic polymers extremely attractive raw materials for scaffold fabrication.
The molecular structures of the most common ones [21, 48, 49] are reported in
Fig. 1.4.
Scientific literature focuses its efforts mainly in the development of scaffolds
made of polyesters, which possess the most suitable features to be employed in
this context. This class of materials can degrade in water as a consequence of
ester bond hydrolysis. It is pointed out, however, that hydrolysis occurs
depending mainly on material hydrophilicity and, for this reason, not all poly-
esters can be considered as hydrolizable materials (the aromatic polyester
polyethylenterephtalate, for example, is highly stable in aqueous environment).
It is worth noting that one potential concern arises from the local pH changes
that can occurs during degradation as a consequence of acid nature of degra-
dation products [50].
Currently, the most widely investigated and most commonly used biomedical
polyesters are polylactide (PLA), polyglycolide (PGA) and their copolymers
(PLAxGAy, where x and y indicate mol% content of the monomers) [51], usually
referred to as poly-a-hydroxyacids (Fig. 1.4). These materials degrade upon water
exposure into products absorbable by the organism: lactic acid, that is normally
produced by muscular contraction, can be metabolized through the citric acid cycle
whereas glycolic acid may be eliminated directly in urine or may be converted to

Fig. 1.4 Common synthetic

biodegradable polymers for
scaffold-based TE
12 1 Introduction

enter the citric acid cycle [52]. Poly-a-hydroxyacids are usually synthesized by
ring-opening polymerization of the cyclic dimers of lactic acid and glycolic acid
(lactide and glycolide). Lactic acid is a chiral molecule existing as L or D isomer,
thus polylactide can be optically pure, poly(L)lactide or poly(D)lactide, or it can
exist in the racemic form, poly(D,L)lactide, depending on the starting monomer.
PGA and the stereoregular forms of PLA are semicrystalline polymers whereas
PLAGA copolymers can be amorphous depending on molecular composition
because the presence of a comonomer disturbs the crystallization ability of the
chains. It has been reported that PLAGA random copolymers with L-lactic acid
units are amorphous when glycolic acid amount falls within the range 25–75%,
whereas PLAGA copolymers with lactic acid in both L and D forms are amorphous
in the range 0–75% of glycolic acid content [53]. Copolymerization modulates not
only the phase morphology (amorphous to crystalline phase ratio) and the thermal
properties (glass transition temperature, crystallization and melting temperature)
but also the degradation rate. Degradation mechanism and kinetics of this series of
polymers have been intensively studied [54, 55]. Hydrolysis rate is primarily
correlated with hydrophobicity of the material that changes depending on the
copolymer composition, being lactic acid more hydrophobic than glycolic acid
because of the presence of an extra CH3 group. The hydrophobicity of lactic acid
limits the water uptake and, accordingly, the homopolymer PGA should be the
faster degrading one, with degradation rate that should decrease with the increase
of lactic acid content. However, hydrophobicity is not the only parameter affecting
degradation rate, which is deeply influenced also by phase morphology. Indeed,
molecular chains packed in the crystalline phase absorb a lower amount of water
with respect to the less dense amorphous phase. This fact explains why the
amorphous PLA50GA50 degrades faster than the semicrystalline PGA, even if
the latter is more hydrophilic [56, 57]. Other factors affecting degradation kinetics
are polymer molecular weight and shape of the device. The possibility to
modulate, within a wide range, degradation rate and mechanical properties by
controlling copolymer composition and molecular structure makes these polyesters
the most widely employed bioresorbable materials suitable for many and different
applications, such as resorbable surgical sutures, drug delivery systems,
orthopaedic appliances and scaffolds.
Polyesters with a long carbon backbone chain are identified as x-polyhy-
droxyalcanoates among which poly(e-caprolactone) (PCL) is frequently employed
to produce scaffolds (Fig. 1.4). Being these polymers highly hydrophobic and
semicrystalline, their degradation kinetic is extremely slow, thus they are suitable
for long-term tissue engineering applications. In particular, e-caprolactone is lar-
gely used also as comonomer to slow down degradation rate of homopolymers
such as PLA (giving rise to PLAxCLy copolymers) [58–60].
Aliphatic polycarbonates are also applied in tissue engineering [61], in par-
ticular poly(1,3-trimethylene carbonate) (PTMC), an elastomeric polymer poten-
tially candidate for soft tissue engineering. Trimethylene carbonate is also widely
used as comonomer with lactic acid, glicolic acid or e-caprolactone in order to
obtain scaffolds with elastomeric properties [62–66].
1.3 The Scaffold 13

Other categories of materials are currently under investigation [67]:

• Polyphosphazenes these polymers with a backbone of alternating phosphorus
and nitrogen atoms are at the interface between inorganic and organic polymers.
Biodegradable polyphosphazenes can be synthesized by incorporating side
groups on phosphorous atoms with the possibility to modulate the degradation
rate over hours, days, months, or years by carefully controlling the nature and
composition of side substitutes [68, 69]. Thanks to their synthetic flexibility,
good biocompatibility, non-toxic degradation products and tailored mechanical
properties they are good candidates for various soft and hard tissue engineering
applications [70–72].
• Poly(propylene fumarate) being available as an injectable system that is cross-
linked in situ, it is a very interesting material for bone tissue application in the
treatment of crevices and defects. Its mechanical properties vary according to
the cross-linking agents used and they are often improved by the addiction of
inorganic particles such as b-TCP [73–75].
• Poly(glycerol sebacate) it is a biodegradable and biocompatible material with
very interesting elastomeric properties that make it a suitable polymer for soft
tissue engineering [76–79].

1.3.2 Scaffold Fabrication Technologies

Selection of the raw material for producing the scaffold is complementary to the
choice of the proper fabrication technology suitable to achieve the desired scaffold
properties. Since TE developed, many techniques have been used to this aim and
even nowadays new methods are invented, though they are mostly modification or
smart combination of already existing techniques. This section aims at providing
an overview of conventional and well-known processes for scaffold fabrication by
illustrating the characteristics of the obtained scaffold [20, 21, 23, 26, 80, 81].
Figure 1.5 reports representative images of scaffolds obtained by the techniques
described in this section.

1.3.2.1 Textile Technologies

Earlier TE scaffolds composed of fibrous biodegradable polymer fabrics were

produced using textile technologies. Mostly PGA and PLA non-woven scaffolds
were used in TE research [89–91]. These fibrous structures have high porosity (up
to 95%) with interconnected pores and high surface area to volume ratio, though
fibre diameter is confined in the range 10–15 lm.
14 1 Introduction

Fig. 1.5 Scanning Electron Microscope images of representative scaffolds obtained by different
fabrication technologies: a textile technologies (reprinted from [21], Copyright (2004), with
permission from Elsevier), b solvent casting and particulate leaching (reprinted from [82],
Copyright (2004), with permission from Elsevier), c freeze drying (reprinted from [83], Copyright
(1999), with permission from Elsevier), d solid freeform fabrication (reprinted from [84],
Copyright (2003), with permission from Elsevier), e thermally induced phase separation (reprinted
from [85], Copyright (1999), with permission from Wiley), f peptide assembly (reprinted from [86],
Copyright (2006), with permission from Elsevier, transmission electron microscopy image), g gas
foaming (reprinted from [87], Copyright (2005), with permission from Wiley), and h electrospin-
ning (reprinted from [88], Copyright (2008), with permission from Elsevier)

1.3.2.2 Solvent Casting and Particulate Leaching

This technique, firstly described by Mikos et al. in 1994 [92], uses a water-soluble
porogen to produce pores within a polymer matrix. In brief, a polymer solution is
cast in a mould containing particles of desired dimension. After solvent evaporation,
particles are leached out by immersion in water. Mainly PLA and PLAGA scaffolds
were produced with this approach. Salts are the most commonly used porogen
[64, 92], but also sugar [93], paraffin [94] and gelatine spheres [94] have been
employed. This method enables to tune independently pore size (up to 500 lm in
diameter) and porosity (up to 90%) by controlling particle dimensions and porogen/
polymer ratio respectively. However, due to gravity, homogeneous pore distribution
is only obtained in scaffolds less than a few millimetres thick [95] and complete
elimination of both organic solvent and porogen is rather difficult to achieve if pores
are not completely interconnected. Moreover, biomolecules potentially added to the
scaffold can be partially removed during the leaching step in water.
1.3 The Scaffold 15

1.3.2.3 Freeze Drying

An emulsion of polymer solution and water is prepared and rapidly cooled down to
block the liquid structure in a solid one. Organic solvent and water are subse-
quently removed by freeze drying leaving a solid porous polymer with porosity up
to 90% [96]. This technique has been applied to many biocompatible polymers
such as PLA, PGA, PLAGA, PCL, chitosan and alginate [97–100]. Despite its
versatility, freeze drying is a time and energy consuming method that takes several
days to completely eliminate solvents.

1.3.2.4 Solid Freeform Fabrication

Solid freeform fabrication process is a computerized technique involving the design

of a scaffold model through a CAD system which elaborates it as a series of cross
sections [84, 101–103]. These sections are built by a rapid prototyping machine that
lays down layers of material starting from the bottom and moving up a layer at a time
to create the scaffold. This method enables to fabricate large and complex 3D objects
with a precise control of pore architecture. Unfortunately, this top-down approach
does not create nano-scaled structures and requires expensive equipments.

1.3.2.5 Thermally Induced Phase Separation

In this approach a polymer solution is cooled to low temperatures in order to

induce a liquid–liquid or solid–liquid phase separation. The solvent located in the
solvent-rich phase is subsequently removed by sublimation, leaving a porous
polymer scaffold [104–106]. Scaffold morphology is controlled by the type of
solvent, polymer concentration and phase separation temperature. This technique
can be used to fabricate scaffolds from many types of polymers and polymeric
composite materials, obtaining highly interconnected pores with tuneable pore size
and also combined macro and microporous structures [107, 108]. The method
enables to produce also nanofibrous networks with porosity of 98% and fibre
diameters in the range 50–500 nm [3].

1.3.2.6 Peptide Self-Assembly

Peptides can be ad hoc synthesized to be able, in certain conditions, to generate

ab-helixes or b-sheets that self-assemble into stable nanofibres [109–111]. The
mechanism is driven by non-covalent bonds and ionic interaction and it is con-
trolled by pH and peptide concentration. These structures are used as synthetic
ECM, even if, due to lack of mechanical strength, they are often used upon
incorporation into more mechanically resilient scaffolds [112].
Two scaffold fabrication technologies, gas foaming and electrospinning, which
were employed in the course of the present Ph.D., are illustrated in more detail in
the following paragraphs.
16 1 Introduction

1.3.2.7 Gas Foaming

Gas foaming has been commonly employed to produce microcellular foams of

thermoplastic polymers [113–116] such as polymethylmethacrylate and polysty-
rene, but only in 1994 Mooney et al. applied this method for the production of
PLA50GA50 scaffolds for TE [117]. Since then gas foaming has become an
appealing technique for fabricating microporous scaffolds [118, 119].
Supercritical carbon dioxide (scCO2) is the most common substance employed,
which, once turned into gas phase, acts as a porogen to generate pores within a
polymer matrix. The method exploits the unique properties of scCO2 that, com-
bining liquid-like densities (high solvent power) with gas-like viscosities (high
diffusion rates) [118], is used for a wide range of applications in polymer syn-
thesis, extraction and impregnation processes, particle formation and blending
[120, 121]. Moreover, CO2 has a relatively low critical point (Tc = 31 C,
Pc = 7.4 MPa) that can be easily achieved in a high-pressure equipped laboratory.
Basically, the method consists in dissolving scCO2 in a solid polymer at high
pressure, generating a low viscosity mixture. Subsequently, the depressurization
decreases scCO2 solubility in the polymer and leads to the phase transition of CO2
from supercritical to gas. CO2 bubble nucleation occurs and nuclei growth generates
pores within the polymer. Concomitantly, viscosity of the polymer-scCO2 mixture
increases till all the gas has escaped from the polymer, leaving behind a solid structure
with ‘‘locked in’’ pores [122] (Fig. 1.6). Scaffold morphology can be controlled by
varying the amount of scCO2 incorporated and its release rate from the polymer.
The main benefit of using scCO2 foaming for the production of scaffolds is the
reduction of problems associated with residual solvents that can be toxic to
mammalian cells. The capability of producing scaffolds without the use of any
toxic solvent makes this technique unique and extremely interesting with respect
to all other scaffold fabrication methods known to date. However, it is well doc-
umented that foamed scaffolds can exhibit inadequate pore interconnectivity
especially at the scaffold surface, where a non-porous layer forms probably
because of the rapid diffusion of the CO2 from the surface as the pressure is
released [123]. This issue has been overcome by combining gas foaming with the

Fig. 1.6 Schematic description of scCO2 foaming process. Polymer is firstly saturated with
scCO2 at high pressure, generating a low viscous polymer-scCO2 mixture. Then, the pressure
release produces a polymer foam
1.3 The Scaffold 17

salt leaching procedure [117, 124] or by trimming off the non-porous skin from the
scaffold after its fabrication [125, 126].
ScCO2 foaming of biodegradable polymers has been mainly applied to poly-
a-hydroxacids, though some publications also report the use of PCL [127, 128].
Given their morphology and their mechanical performances, these scaffolds can be
employed for TE of hard tissues, such as bone and cartilage. For such applications
PLA e PLAGA copolymers with different compositions have been successfully
foamed either as plain materials [117, 125, 129] or in combination with inorganic
substances [87, 130]. Moreover, scCO2 foaming is a powerful technology for the
production of scaffolds loaded with biomolecules such as growth factors [131–135]
and DNA [134, 135] that, once released, have been shown to maintain their activity.

1.3.2.8 Electrospinning

Electrospinning (ES) enables the production of non-woven mats composed of sub-

micrometric fibres from a polymer solution or melt. The process was invented at
the beginning of 1900 by Cooley and Morton [136, 137] and it was mainly
developed thanks to the contribution of Formhals’ patents [138–142] for textile
applications. However, only at the beginning of this century ES began to be used
for scaffold fabrication [143–147] and nowadays it is considered a powerful
technique that is employed to this aim by many researchers all around the world.
ES (Fig. 1.7) involves the application of a high voltage difference between a
positively charged metallic needle, ejecting the polymeric solution, and a groun-
ded collector. When the electrostatic force overcomes the cohesive force of the
solution an electrically charged jet emerges from the capillary. The fluid jet is
accelerated and stretched by the external electric field and thins dramatically while
travelling towards the collector, leading to the formation of continuous solid fibres
as the solvent evaporates. ES products appear as highly porous non-woven sheets
made of submicrometric fibres with large surface area to volume ratio. Fibre
diameter, morphology and arrangement in space can be controlled by a proper
selection of the spinning parameters.
ES has been demonstrated to be an extremely versatile technology able to
produce fibres with diameters ranging from few nanometers to several microns,
from over two hundred synthetic and natural polymers, in the form of plain fibres,
blends and organic–inorganic composite fibres [148–150]. Another advantage is
the simplicity of the process that does not require any sophisticated and expensive
equipment and that can be easily scaled-up for mass production.
Besides the aforementioned benefits of the process, its power arises from the
morphological features of the products obtained. Indeed, ES fibre dimensions and
spatial organization resemble the fibrous component of ECM, making ES a
technology for the production of morphologically biomimetic scaffolds. As a
consequence, this kind of scaffolds can be used to elicit different responses from
the same cell phenotype only thanks to their particular topography. Indeed, it is
well-established that, besides being influenced by external chemical signals
18 1 Introduction

Fig. 1.7 Scheme of the ES

process

coming both from ECM and from nearby cells, cell behaviour is manipulated also
by the morphological features of their environment that control cell adhesion,
orientation, motility, gene expression, etc. [151]. A comprehensive review about
the effect of surface topography on cells is provided by Stevens et al. [152]. The
authors describe cell behaviour interacting with differently structured scaffolds and
conclude that nanoscaled architectures promote better spreading and attachment
when compared with microscaled scaffolds (Fig. 1.8). Their model was supported
by several studies reporting that cells are able to better adhere and spread when
cultured on sub-micrometric fibres with respect to micrometric ones [153, 154].
Given the many advantages in using ES technology, this is certainly one of the
most extensively used techniques for scaffold fabrication. The scientific literature
reports many successful applications of ES products for supporting different types
of cells, demonstrating that these kinds of scaffolds are promising for the regen-
eration of several types of tissues (Table 1.2). Given the intrinsic mechanical
properties of ES non-woven scaffolds, they are mainly applied for the recon-
struction of soft tissues. As an example, the non-woven nanofibrous sheets
obtained by ES are suitable for wound dressing and wound healing applications.
The process also allows to obtain highly aligned fibres that are able to support and
direct cells which are oriented in their natural environment, such as neuronal cells
[155]. Moreover, thanks to the versatility of the technology, tubular scaffolds,
which are ideally suited for replacing damaged blood vessels, can be easily fab-
ricated [156]. ES scaffolds are also studied for hard TE (e.g. bone) even if, for
these applications, polymeric materials are usually combined with inorganic
substances in order to improve the biomimetic and mechanical features of the
scaffold [157].
The high surface area to volume ratio of ES mats makes them also suitable for
drug delivery applications. Many substances have been incorporated within fibres
1.3 The Scaffold 19

Fig. 1.8 Cells binding to

microstructured scaffolds
flatten, exhibiting a
morphology similar to that
assumed on flat surfaces
(such as 2D TCPS). Instead,
nanostructured scaffolds
offers many binding sites to
cell membrane receptors and
allow cells to assume a spread
morphology more similar to
that they have in natural ECM
(figure adapted from [152],
Copyright (2005), with
permission from AAAS
publications)

Table 1.2 Examples of ES TE Application Materials References

biomaterials designed for TE
applications Skin PCL/collagen [158, 159]
Collagen [156]
Chitin [154]
Polystyrene [160]
Polyurethane [161]
Nerve PCL [162, 163]
PCL/collagen [164]
PLA [165, 166]
PLA10GA90 [167]
Bone PCL [168]
PCL/b-TCP [169]
PCL/CaCO3 [170]
PLA/HA [171]
Gelatin/HA [172]
Vascular graft PCL/collagen/elastin [173]
PLA/collagen/elastin [173, 174]
PLA80CL20/collagen/elastin [173]
PLA50GA50/collagen/elastin [173, 175]
PLA75CL25 [59]
PLA50CL50 [176]
Collagen/eleastin [177]
PLA/PCL [178]
PLA75GA25/collagen [179]
Silk [180]
Heart PCL [181]
Pulyurethane [182]
PLA/PLA75GA25/PLA10GA90 [183]
Cartilage PCL [184]
PCL/PLA [185]
20 1 Introduction

simply by adding them to polymer solution or through coaxial ES [186, 187].

The latter process consists in electrospinning two different solutions flowing
through two capillaries, with the smaller one positioned inside the larger one. This
configuration can encapsulate a smaller fibre within a larger one, leading to core–
shell morphology. Drugs [188–192], proteins [193–197], growth factors [198, 199]
and DNA [200] have been successfully incorporated inside polymeric ES fibres. It
is worth noting that the capability to incorporate biologically relevant substances
within scaffolds in a one-step process gives the possibility to combine, in a unique
product, the function of supporting cell growth with the beneficial effects that arise
from the interaction of cells with the delivered biomolecules.

1.4 Scaffold Functionalization

The need to address cell activity towards tissue regeneration has driven the
scientific community to make efforts for designing bio-functionalized scaffolds.
The main objective is to make the scaffold as much biomimetic as possible, not
only in terms of 3D structural features, but also from a chemical point of view.
To this aim the ECM is inevitably taken as a benchmark.
As previously discussed in Sect. 1.2, ECM and the hosted cells communicate
through specific proteins either located at the surface of the ECM fibrous com-
ponent (e.g. laminin, fibronectin, vitronectin, etc.) or dissolved in the gel-like ECM
component (e.g. growth factors). The incorporation of these proteins (or of pep-
tides displaying similar functionalities) is considered a valid approach to confer
bioactivity to the scaffold and it is commonly achieved through either bulk or
surface functionalization. In the bulk functionalization, biomolecules are incor-
porated within the polymer matrix and they are released in the surrounding
environment either by diffusion or via scaffold degradation. Growth factors (GFs)
are probably the most widely used biomolecules in such applications [201]; the
role of these proteins is to transmit signals controlling cell migration, differenti-
ation, and proliferation. A list of the most commonly employed GFs together with
their known activities is provided by Chen and Mooney [202]. Other molecules
that have been incorporated within scaffold matrixes are antibiotics, anticancer
drugs, anti-inflammatory dugs and DNA plasmids [203].
Surface modification is the other approach aimed at conferring bioactivity to
scaffolds since biomaterial surface properties regulate the interaction with the
physiological environment and with cells. In particular, the scientific community
has dedicated strong efforts in modifying biomaterial to stimulate cell adhesion by
binding specific peptides sequences to biomaterial surface. RGD sequence
(R: arginine; G: glycine; D: aspartic acid) has been largely employed for stimu-
lating cell attachment on synthetic surfaces since it was identified as the minimal
sequence required to mediate cell adhesion in most ECM proteins. RGD can be
introduced in the biomaterial either by synthesizing polymers containing RGD
sequences in the macromolecular chains [204] or via post-fabrication treatments
1.4 Scaffold Functionalization 21

for attaching RGD sequences to material surfaces. In this context, films have been
mostly employed whereas modification of scaffold surface with these biomole-
cules is less explored [204].
In the course of the present Ph.D. both fuctionalization approaches were
attempted. Bulk functionalization was achieved through the incorporation of a GF
in ES fibres whereas surface functionalization of ES fibres was carried out by
Surface Initiated Atom Transfer Radical Polymerization (SI-ATRP).

References

1. Mason C, Dunnill P (2008) A brief definition of regenerative medicine. Regen Med 3:1
2. Vacanti JP, Vacanti CA (2007) The history and scope of tissue engineering. In: Lanza R,
Langer R, Vacanti J (eds) Principles of Tissue Engineering. Elsevier, San Diego, pp 3–6
3. Langer R, Vacanti JP (1993) Tissue engineering. Science 260:920
4. Leor J, Amsalem Y, Cohen S (2005) Cells, scaffolds and molecules for myocardial tissue
engineering. Pharmacol Ther 105:151
5. Stock UA, Vacanti JP (2001) Tissue engineering: current state and prospects. Ann Rev Med
52:443
6. Place ES, Evans ND, Stevens MM (2009) Complexity in biomaterials for tissue
engineering. Nat Mater 8:457
7. Slack J (2007) Molecular biology of the cell. In: Lanza R, Langer R, Vacanti J (eds)
Principles of Tissue Engineering. Elsevier, San Diego, pp 53–66
8. Bilodeau K, Mantovani D (2006) Bioreactors for tissue engineering: focus on mechanical
constrains. A comparative review. Tissue Eng 12:2367
9. Vunjak-Novakovic G, Freed LE, Biron RJ, Langer R (1996) Effects of mixing on the
composition and morphology of tissue-engineered cartilage. Bioengineering Food Nat Prod
42:850
10. Martin I, Wendt D, Heberer M (2004) The role of bioreactors in tissue engineering. Trends
Biotechnol 22:80
11. Folkman J, Moscona A (1978) Role of cell shape in growth control. Nature 273:345
12. Lutolf MP, Hubbell JA (2005) Synthetic biomaterials as instructive extracellular
microenvironments for morphogenesis in tissue engineering. Nat Biotechnol 23:47
13. Giancotti FG (2000) Complexity and specificity of integrin signaling. Nat Cell Biol 2:E13
14. Bissell MJ, Hall HG, Parry G (1982) How does the extracellular matrix direct gene
expression? J Theor Biol 99:31
15. Streuli CH, Bailey N, Bissell MJ (1991) Control of mammary epithelial differentiation:
basement membrane induces tissue-specific gene expression in the absence of cell-cell
interaction and morphological polarity. J Cell Biol 115:1383
16. Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE (1997) Geometric control of
cell live and death. Science 276:1425
17. Blaschke RJ, Howlett AR, Desprez P-Y, Peterson OW, Bissell MJ (1994) Cell
differentiation by extracellular matrix components. Methods Enzymol 245:535
18. Veiseh M, Turley EA, Bissell MJ (2008) Top–down analysis of a dynamic environment:
extracellular matrix structure and function. In: Laurencin CT, Nair LS (eds) Nanotechnology
and Tissue Engineering: The Scaffold. CRC Press/Taylor & Francis Group, Boca Raton,
pp 33–51
19. ASTM F2150-07 (2007) Standard guide for characterization and testing of biomaterial
scaffolds used in tissue-engineered medical products
20. Salgado AJ, Coutinho OP, Reis RL (2004) Bone tissue engineering: state of the art and
future trends. Macromol Biosci 4:743
22 1 Introduction

21. Ma PX (2004) Scaffolds for tissue fabrication. Materials Today 7:30

22. Yoon DM, Fisher JP (2007) Polymeric scaffolds for tissue engineering applications.
In: Fisher JP, Mikos AG, Bronzino JD (eds) Tissue Engineering. CRC Press/Taylor &
Francis Group, Boca Raton, pp 8-1–8-18
23. Karande TS, Agrawal CM (2008) Functions and requirments of synthetic scaffolds in tissue
engineering. In: Laurencin CT, Nair LS (eds) Nanotechnology and Tissue Engineering: The
Scaffold. CRC Press/Taylor & Francis Group, Boca Raton, pp 53–86
24. MUschler GF, Nakamoto C, Griffith LG (2004) Engineering principles of clinical cell-based
tissue engineering. J Bone Joint Surg 86-A:1541
25. Vert M, Li SM, Spenlehauer G, Guerin P (1992) Bioresorbability and biocompatibility of
aliphatic polyesters. J Mater Sci Mater Med 3:432
26. Hutmacher DW (2000) Scaffolds in tissue engineering bone and cartilage. Biomaterials
21:2529
27. Muschler GF, Nakamoto C, Griffith LG (2004) Engineering principles of clinical cell-based
tissue engineering. J Bone Joint Surg 86A:1541
28. Van der Flier A, Sonnenberg A (2001) Function interactions of integrins. Cell Tissue Res
305:285
29. Plow EF, Haas TA, Zhang L, Loftus J, Smith JW (2000) Ligand binding to integrins. J Biol
Chem 275:21785
30. Roach P, Farrar D, Perry CC (2005) Interpretation of protein adsorption: surface-induced
conformational changes. J Am Chem Soc 127:8168
31. Mathieu HJ (2001) Bioengineered material sufaces for medical applications. Surf Interface
Anal 32:3
32. Saltzman WM, Kyriakides TR (2007) Cell interactions with polymers. In: Lanza R, Langer
R, Vacanti J (eds) Principles of Tissue Engineering. Elsevier, San Diego, pp 279–296
33. Jiao Y-P, Cui F-Z (2007) Surface modification of polyester biomaterials for tissue
engineering. Biomed Mater 2:R24
34. Karande TS, Ong JL, Agrawal CM (2004) Diffusion in musculoskeletal tissue engineering
scaffolds: design issues related to porosity, permeability, architecture, and nutrient mixing.
Ann Biomed Eng 32:1728
35. Kim B-S, Mooney DJ (1998) Development of biocompatible synthetic extracellular
matrices for tissue engineering. Trends Biotechnol 16:224
36. Williams DF (1987) Definitions in biomaterials. In: Proceeding of a Consensus Conference
of the European Society for Biomaterials. Elsevier, Amsterdam
37. Park JB (2000) Biomaterials. In: Bronzino JD (ed) The Biomedical Engineering Handbook.
CRC Press LLC, Boca Raton, p IV-1
38. Hench LL, Polak JM (2002) Third-generation biomedical materials. Science 295:1014
39. Kim S-S, Park MS, Jeon O, Choi CY, Kim B-S (2006) Poly(lactide-co-glycolide)/
hydroxyapatite composite scaffolds for bone tissue engineering. Biomaterials 27:1399
40. Zhang R, Ma PX (1999) Poly(a-hydroxyl acids)/hydroxyapatite porous composites for
bone-tissue engineering. I. Preparation and morphology. J Biomed Mater Res 44:446
41. Rezwan K, Chen QZ, Blacker JJ, Boccaccini AR (2006) Biodegradable and bioactive
porous polymer/inorganic composite scaffolds for bone tissue engineering. Biomaterials
27:3413
42. Ikada Y (2006) Tissue engineering: fundamentals and applications. Elsevier, Amsterdam
43. Caterson EJ, Nesti LJ, Li W-J, Danielson KG, Albert TJ, Vaccaro AR, Tuan RS (2001)
Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/
alginate amalgam. J Biomed Mater Res 57:394
44. Zhang Y, Zhang M (2001) Synthesis and characterization of macroporous chitosan/calcium
phosphate composite scaffolds for tissue engineering. J Biomed Mater Res 55:304
45. Kim H-W, Kim H-E, Salih V (2005) Stimulation of osteoblast responses to biomimetic
nanocomposites of gelatin-hydroxyapatite for tissue engineering scaffolds. Biomaterials
26:5221
References 23

46. Chen G, Sato T, Ushida T, Hirochika R, Shirasaki Y, Ochiai N, Tateishi T (2003) The use of
a novel PLGA fiber/collagen composite web as a scaffold for engineering of articular
cartilage tissue with adjustable thikness. J Biomed Mater Res 67:1170
47. Sarasam AR, Samli AI, Hess L, IHnat MA, Madihally SV (2007) Blending chitosan with
polycaprolactone: porous scaffolds and toxicity. Macromol Biosci 7:1160
48. Agrawal CM, Ray RB (2001) Biodegradable polymeric scaffolds for musculoskeletal tissue
engineering. J Biomed Mater Res 55:141
49. Seal BL, Otero TC, Panitch A (2001) Polymeric biomaterials for tissue and organ
regeneration. Mater Sci Eng R 34:147
50. Agrawal CM, Athanasiou KA (1997) Technique to control pH in vicinity of biodegrading
PLA-PGA implants. J Biomed Mater Res 38:105
51. Morita S-I, Ikada Y (2002) Lactide copolymers for scaffolds in tissue engineering.
In: Tissue engineering and biodegradable equivalents: scientific and clinical applications,
pp 111–122
52. Athanasiou KA, Agrawal CM, Barber FA, Burkhart SS (1998) Orthopaedic applications for
PLA-PGA biodegradable polymers. Arthrosc J Arthrosc Relat Surg 14:726
53. Gilding DK, Reed AM (1979) Biodegradable polymers for use in surgery–polyglycolic/
poly(actic acid) homo- and copolymers. Polymer 20:1459
54. Li S (1999) Hydrolytic degradation characteristics of aliphatic polyesters derived from
lactic and glycolic acid. J Biomed Mater Res 48:342
55. Vert M, Garreau H, Maudit J, Boustta M, Schwach G, Engel R, Coudane J (1997)
Complexity of the hydrolitic degradation of aliphatic polyesters. Die Angew Makrom Chem
247:239
56. Li S, Garreau H, Vert M (1990) Structure-property relationships in the case of the
degradation of massive aliphatic poly-(a-hydroxy acids) in aqueous media, part
2:degradation of lactide-glycolide copolymers: PLA37.5GA25 and PLA75GA25. J Mater
Sci Mater Med 1:131
57. Miller RA, Brady JM, Cutright DE (1977) Degradation rate of oral resorbable implants
(polylactate and polyglycolate): rate modification with changes in PLA/PGA copolymer
ratios. J Biomed Mater Res 11:711
58. De Groot JH, Zijlstra FM, Kuipers HW, Pennings AJ, Klompmaker J, Veth RPH, Jansen
HWB (1997) Meniscal tissue regeneration in porous 50/50 copoly(L-lactide/e-caprolactone)
implants. Biomaterials 18:613
59. Xu CY, Inai R, Kotaki M, Ramakrishna S (2004) Aligned biodegradable nanofibrous
structure: a potential scaffold for blood vessel engineering. Biomaterials 25:877
60. Mo XM, Xu CY, Kotaki M, Ramakrishna S (2004) Electrospun P(LLA-CL) nanofiber:
a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation.
Biomaterials 25:1883
61. Welle A, Kroger M, Doring M, Niederer K, Pindel E, Chronakis IS (2007) Electrospun
aliphatic polycarbonates as tailored tissue scaffold materials. Biomaterials 28:2211
62. Mukherjee DP, Smith DF, Rogers SH, Emmanual JE, Jadin KD, Hayes BK (2009) Effect of
3D-microstructure of bioabsorbable PGA:TMC scaffolds on the growth of chondrogenic
cell. J Biomed Mater Res Part B Appl Biomater 88B:92
63. Vinoy T, Zhang X, Catledge SA, Vohra YK (2007) Functionally graded electrospun
scaffolds with tunable mechanical properties for vascular tissue regeneration. Biomed Mater
2:224
64. Pego AP, Siebum B, Luyn V, Gallego XJ, Seijen YV, Poot AA, Grijpma DW, Feijen J
(2003) Preparation of degradable porous structures based on 1, 3-trimethylene carbonate
and D, L-lactide (co)polymers for heart tissue engineering. Tissue Eng 9:981
65. Pego AP, Poot AA, Grijpma DW, Feijen J (2003) Biodegradable elastomeric scaffolds for
soft tissue engineering. J Controlled Release 87:69
66. Plikk P, Malberg S, Albertsson A-C (2009) Design of resorbable porous tubular copolyester
scaffolds for use in nerve regeneration. Biomacromolecules 10:1259
24 1 Introduction

67. Martina M, Hutmacher DW (2007) Biodegradable polymers applied in tissue engineering

research: a review. Polym Int 56:145
68. Ambrosio AM, Allcock HR, Katti DS, Laurencin CT (2002) Degradable polyphosphazene/
poly(hydroxyester) blends: degradation studies. Biomaterials 23:1667
69. Allcock HR, Fuller TJ, Matsumura K (1982) Hydrolysis pathways for amminophosphazenes.
Inorg Chem 21:515
70. Laurencin CT, El-Amin SF, Ibim SE, Willoughby DA, Attawia M, Allcock HR, Ambrosio
AM (1996) A highly porous 3-dimensional polyphosphazene polymer matrix for skeletal
tissue regeneration. J Biomed Mater Res 30:133
71. Conconi MT, Lora S, Baiguera S, Boscolo E, Folin M, Scienza R, Rebuffat P, Parnigotto
PP, Nussdorfer GG (2004) In vitro culture of rat neuromicrovascular endothelial cells on
polymeric scaffolds. J Biomed Mater Res 71A:669
72. Ambrosio AM, Sahota JS, Runge C, Kurtz SM, Lakshmi S, Allcock HR, Laurencin CT
(2003) Novel polyphosphazene-hydroxyapatite composites as biomaterials. IEEE Eng Med
Biol Mag 22:18
73. Temenoff JS, Mikos AG (2000) Injectable biodegradable materials for orthopedic tissue
engineering. Biomaterials 21:2405
74. Payne RG, McGonigle JS, Yaszemski Mj, Yasko AW, Mikos AG (2002) Development of
an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell
populations Part 3 Proliferation, differentiation of encapsulated marrow stromal osteoblasts
cultured on crosslinking poly(propylene fumarate). Biomaterials 23:4381
75. Yaszemski Mj, Payne RG, Hayes WC, Langer R, Aufdemorte TB, Mikos AG (1995) The
ingrowth of new bone tissue, initial mechanical properties of a degrading polymeric
composite scaffold. Tissue Eng 1:41
76. Sundback CA, Shyu JY, Wang Y, Faquin WC, Langer R, Vacanti J, Hadlock TA (2005)
Biocompatibility analysis of poly(glycerol sebacate) as a nerve guide material. Biomaterials
26:5454
77. Gao J, Ensley AE, Nerem RM, Wang Y (2007) Poly(glycerol sebacate) supports the
proliferation and phenotypic protein expression of primary baboon vascular cells. J Biomed
Mater Res 83A:1070
78. Gao J, Crapo PM, Wang Y (2006) Macroporous elastomeric scaffolds with extensive
micropores for soft tissue engineering. Tissue Eng 12:917
79. Wang Y, Ameer GA, Sheppard BJ, Langer R (2002) A tough biodegradable elastomer. Nat
Biotechnol 20:602
80. Smith LA, Ma PX (2004) Nano-fibrous scaffolds for tissue engineering. Colloids Surf B
Biointerfaces 39:125
81. Murphy MB, Mikos AG (2007) Polymer scaffold fabrication. In: Lanza R, Langer R,
Vacanti J (eds) Principles of Tissue Engineering. Elsevier, San Diego, pp 309–321
82. Katoh K, Tanabe T, Yamauchi K (2004) Novel approach to fabricate keratin sponge
scaffolds with controlled pore size and porosity. Biomaterials 25:4255
83. Kang HW, Tabata Y, Ikada Y (1999) Fabrication of porous gelatin scaffolds for tissue
engineering. Biomaterials 20:1339
84. Leong KF, Cheah CM, Chua CK (2003) Solid freeform fabrication of three-dimensional
scaffolds for engineering replacement tissues and organs. Biomaterials 24:2363
85. Ma PX, Zhang R (1999) Synthetic nano-scale fibrous extracellular matrix. J Biomed Mater
Res 46:60
86. Woolfson DN, Ryadnov MG (2006) Peptide-based fibrous biomaterials: some things old,
new and borrowed. Curr Opin Chem Biol 10:559
87. Mathieu L, Montjovent M-O, Bourban P-E, Pioletti DP, Manson J-AE (2005) Bioresorbable
composites prepared by supercritical fluid foaming. J Biomed Mater Res Part A 75:89
88. Ma K, Chan CK, Liao S, Hwang WYK, Feng Q, Ramakrishna S (2008) Electrospun
nanofiber scaffolds for rapid and rich capture of bone marrow-derived hematopoietic stem
cells. Biomaterials 29:2096
References 25

89. Grande DA, Halberstadt C, Naughton G, Schwartz R, Manji R (1997) Evaluation of matrix
scaffolds for tissue engineering of articular cartilage grafts. J Biomed Mater Res 34:211
90. Sittinger M, Reitzel D, Dauner M, Hierlemann H, Hammer C, Kastenbauer E, Planck H,
Burmester GR, Bujia J (1996) Resorbable polyesters in cartilage engineering: affinity and
biocompatibility of polymer fiber structures to chondrocytes. J Biomed Mater Res Part B
Appl Biomater 33:57
91. Ma PX, Langer R (1999) Morphology and mechanical function of long-term in vitro
engineered cartilage. J Biomed Mater Res 44:217
92. Mikos AG, Thorsen AJ, Czerwonka LA, Bao Y, Langer R, Winslow DN, Vacanti JP (1994)
Preparation and characterization of poly(-lactic acid) foams. Polymer 35:1068
93. Holy CE, Dang SM, Davies JE, Shoichet MS (1999) In vitro degradation of a novel
poly(lactide-co-glycolide) 75/25 foam. Biomaterials 20:1177
94. Suh SW, Shin YJ, Kim J, Min CH, Beak CH, Kim D-I, Kim H, Jeon SS, Choo IW (2002)
Effect of different particles on cell proliferation in polymer scaffolds using a solvent-casting
and particulate leaching technique. ASAIO J 48:460
95. Wake MC, Gupta PK, Mikos AG (1996) Fabrication of pliable biodegradable polymer
foams to engineer soft tissues. Cell Transplant 5:465
96. Whang K, Thomas H, Healy KE, Nuber G (1995) A novel method to fabricate
bioabsorbable scaffolds. Polymer 36:837
97. Whang K, Goldstick TK, Healy KE (2000) A biodegradable polymer scaffold for delivery of
osteotropic factors. Biomaterials 21:2545
98. Shen F, Cui YL, Yang LF, Yao KD, Dong XH, Jia WY, Shi HD (2000) A study on the
fabrication of porous chitosan/gelatin network scaffold for tissue engineering. Polym Int
49:1596
99. Hou Q, Grijpma DW, Feijen J (2003) Preparation of interconnected highly porous
polymeric structures by a replication and freeze-drying process. J Biomed Mater Res Part B
Appl Biomater 67B:732
100. Ho M-H, Kuo P-Y, Hsieh H-J, Hsien T-Y, Hou L-T, Lai J-Y, Wang D-M (2004) Preparation
of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterials
25:129
101. Hollister SJ (2005) Porous scaffold design for tissue engineering. Nat Mater 4:518
102. Yan Y, Xiong Z, Hu Y, Wang S, Zhang R, Zhang C (2003) Layered manufacturing of tissue
engineering scaffolds via multi-nozzle deposition. Mater Lett 57:2623
103. Hutmacher DW, Sittinger M, Risbud MV (2004) Scaffold-based tissue engineering:
rationale for computer-aided design and solid free-form fabrication systems. Trends
Biotechnol 22:354
104. Lo H, Kadiyala S, Guggino SE, Leong KW (1996) Poly(L-lactic acid) foams with cell
seeding and controlled-release capacity. J Biomed Mater Res 30:475
105. Blaker JJ, Maquet V, Jerome R, Boccaccini AR, Nazhat SN (2005) Mechanical properties
of highly porous PDLLA/Bioglass composite foams as scaffolds for bone tissue engineering.
Acta Biomater 1:643
106. Schugens C, Maquet V, Grandfils C, Jerome R, Teyssie P (1996) Polylactide macroporous
biodegradable implants for cell transplantation. II. Preparation of polylactide foams by
liquid-liquid phase separation. J Biomed Mater Res 30:449
107. Nam YS, Park TG (1999) Porous biodegradable polymeric scaffolds prepared by thermally
induced phase separation. J Biomed Mater Res 47:8
108. Nam YS, Park TG (1999) Biodegradable polymeric microcellular foams by modified
thermally induced phase separation method. Biomaterials 20:1783
109. Holmes TC (2002) Novel peptide-based biomaterial scaffolds for tissue engineering. Trends
Biotechnol 20:16
110. Beniash E, Hartgerink JD, Storrie H, Stendhal JC, Stupp SI (2005) Self-assembling peptide
amphiphile nanofiber matrices for cell entrapment. Acta Biomater 1:387
111. Yokoi H, Kinoshita T, Zhang S (2005) Dynamic reassembly of peptide RADA16 nanofiber
scaffold. Proc Nat Acad Sci U S A 102:8414
26 1 Introduction

112. Harrington DA, Cheng EY, Guler MO, Lee LK, Donovan JL, Claussen RC, Stupp SI (2006)
Branched peptide-amphiphiles as self-assembling coatings for tissue engineering scaffolds.
J Biomed Mater Res 78A:157
113. Goel SK, Beckman EJ (1994) Generation of microcellular polymeric foams using
supercritical carbon dioxide. I: effect of pressure and temperature on nucleation. Polym
Eng Sci 34:1137
114. Arora KA, Lesser AJ, McCarthy TJ (1998) Preparation and characterization of microcellular
polystyrene foams processed in supercritical carbon dioxide. Macromolecules 31:4614
115. Colton JS, Suh NP (1987) The nucleation of microcellular thermoplastic foam with
additives part I: theoretical considerations. Polym Eng Sci 27:485
116. Kumar V, Suh NP (1990) A process for making microcellular thermoplastic parts. Polym
Eng Sci 30:1323
117. Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R (1996) Novel approach to fabricate
porous sponges of poly(D, L-lactic-co-glycolic acid) without the use of organic solvents.
Biomaterials 17:1417
118. Woods HM, Silva MCG, Nouvel C, Shakesheff KM, Howdle SM (2004) Materials
processing in supercritical carbon dioxide: surfactants, polymers and biomaterials. J Mater
Chem 14:1663
119. Quirk RA, France RM, Shakesheff KM, Howdle SM (2004) Supercritical fluid technologies
and tissue engineering scaffolds. Curr Opinion Solid State Mater Sci 8:313
120. Tomasko DL, Li H, Liu D, Han X, Wingert MJ, Lee LJ, Koelling KW (2003) A review of
CO2 applications in the processing of polymers. Ind Eng Chem Res 42:6431
121. Kazarian SG (2000) Polymer processing with supercritical fluids. Polym Sci Ser C 42:78
122. Barry JJA, Silva MMCG, Popov VK, Shakesheff KM, Howdle SM (2006) Supercritical
carbon dioxide: putting the fizz into biomaterials. Philos Trans Roy Soc A 364:249
123. Goel SK, Beckman EJ (1994) Generation of microcellular polymeric foams using
supercritical carbon dioxide. II: cell growth and skin formation. Polym Eng Sci 34:1148
124. Murphy WL, Dennis RG, Kileny JL, Mooney DJ (2002) Salt fusion: an approach to improve
pore interconnectivity within tissue engineering scaffolds. Tissue Eng 8:43
125. Tai H, Mather ML, Howard D, Wang W, White LJ, Crowe JA, Morgan SP, Williams DJ,
Howdle SM, Shakesheff KM (2007) Control of pore size and structure of tissue engineering
scaffolds produced by supercritical fluid processing. Eur Cell Mater 14:64
126. Barry JJA, Gidda HS, Scotchford CA, Howdle SM (2004) Porous methacrylate scaffolds:
supercritical fluid fabrication and in vitro chondrocyte responses. Biomaterials 25:3559
127. Jenkins MJ, Harrison KL, Silva MCG, Whitaker MJ, Shakesheff KM, Howdle SM (2006)
Characterization of microcellular foams produced from semi-crystalline PCL using
supercritical carbon dioxide. Eur Polym J 42:3145
128. Xu Q, Ren X, Chang Y, Wang J, Yu L, Dean K (2004) Generation of microcellular
biodegradable polycaprolactone foams in supercritical carbon dioxide. J Appl Polym Sci
94:593
129. Singh L, Kumar V, Ratner BD (2004) Generation of porous microcellular 85/15 poly
(dl-lactide-co-glycolide) foams for biomedical applications. Biomaterials 25:2611
130. Mathieu LM, Mueller TL, Bourban P-E, Pioletti DP, Muller R, Manson J-AE (2006)
Architecture and properties of anisotropic polymer composite scaffolds for bone tissue
engineering. Biomaterials 27:916
131. Sheridan MH, Shea LD, Peters MC, Mooney DJ (2000) Bioabsorbable polymer scaffolds for
tissue engineering capable of sustained growth factor delivery. J Controlled Release 64:91
132. Yang XB, Whitaker MJ, Sebald W, Clarke N, Howdle SM, Shakesheff KM, Oreffo ROC
(2004) Human osteoprogenitor bone formation using encapsulated bone morphogenetic
protein 2 in porous polymer scaffolds. Tissue Eng 10:1037
133. Hile DD, Amipour L, Akgerman A, Pishko MV (2000) Active growth factor delivery from
poly(D, L-lactide-co-glycolide) foams prepared in supercritical CO2. J Controlled Release
66:177
References 27

134. Heyde M, Partridge KA, Howdle SM, Oreffo ROC, Garnett MC, Shakesheff KM (2007)
Development of a slow non-viral DNA release system from PDLLA scaffolds fabricated
using a supercritical CO2 technique. Biotechnol Bioeng 98:679
135. Jang J-H, Shea LD (2003) Controllable delivery of non-viral DNA from porous scaffolds.
J Controlled Release 86:157
136. Cooley JF (1902) Apparatus for electrically dispersing fluids. US patent 692631
137. Morton WJ (1902) Method of dispersing fluids. US patent 705691
138. Formhals A (1934) Process and apparatus for preparing artificial threads. US patent
1975504
139. Formhals A (1939) Method and apparatus for spinning. US patent 2160962
140. Formhals A (1940) Artificial thread and method of producing same. US patent 2187306
141. Formhals A (1943) Production of artificial fibers from fiber forming liquids. US patent
2323025
142. Formhals A (1944) Method and apparatus for spinning. US patent 2349950
143. Boland ED, Wnek GE, Simpson DG, Pawlowski KJ, Bowlin G (2001) Tailoring tissue
engineering scaffolds using electrostatic processing techniques: a study of poly(glygolic
acid) electrospinning. J Macromol Sci Pure Appl Chem A38:1231
144. Matthews JA, Wnek GE, Simpson DG, Bowlin GL (2002) Electrospinning of collagen
nanofibers. Biomacromolecules 3:232
145. Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK (2002) Electrospun nanofibrous
structure: a novel scaffold for tissue engineering. J Biomed Mater Res 60:613
146. Boland ED, Bowlin GL, Simpson DG, Wnek GE (2001) Electrospinning of tissue
engineering scaffolds. Polym Mater Sci Eng 85:51
147. Huang L, Nagapudi K, Apkarian RP, Chaikof EL (2001) Engineered collagen-PEO
nanofibers and fabrics. J Biomater Sci Polym Edition 12:979
148. Liang D, Hsiao BS, Chu B (2007) Functional electrospun nanofibrous scaffolds for
biomedical applications. Adv Drug Deliv Rev 59:1392
149. Chew SY, Wen Y, Dzenis Y, Leong KW (2006) The role of electrospinning in the energing
field of nanomedicine. Curr Pharm Des 12:4751
150. Shiffman JD, Schauer CL (2008) A review: electrospinning of biopolymer nanofibres and
their applications. Polym Rev 48:317
151. Webster TJ, Schadler LS, Siegel RW, Bizios R (2001) Mechanisms of enhanced osteoblast
adhesion on nanophase alumina involve vitronectin. Tissue Eng 7:291
152. Stevens MM, George JH (2005) Exploring and engineering the cell surface interface.
Science 310:1135
153. Kwon IK, Kidoaki S, Matsuda T (2005) Electrospun nano- to microfiber fabrics made of
biodegradable copolyesters: structural characteristics, mechanical properties and cell
adhesion potential. Biomaterials 26:3929
154. Noh HK, Lee SW, Kim JM, Oh JE, Kim KH, Chung CP, Choi SC, Park WH, Min BM
(2006) Electrospinning of chitin nanofibers: degradation behavior and cellular response to
normal human keratinocytes and fibroblasts. Biomaterials 27:3934
155. Cao H, Liu T, Chew SY (2009) The application of nanofibrous scaffolds in neural tissue
engineering. Adv Drug Deliv Rev 61:1055
156. Sell SA, McClure MJ, Garg K, Wolfe PS, Bowlin GL (2009) Electrospinning of collagen/
biopolymers for regenerative medicine and cardiovascular tissue engineering. Adv Drug
Deliv Rev 61:1007
157. Jang JH, Castano O, Kim HW (2009) Electrospun materials as potential platforms for bone
tissue engineering. Adv Drug Deliv Rev 61:1065
158. Chong EJ, Phan TT, Lim IJ, Zhang YZ, Bay BH, Ramakrishna S, Lim CT (2007)
Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered
dermal reconstruction. Acta Biomater 3:321
159. Venugopal JR, Zhang Y, Ramakrishna S (2006) In vitro culture of human dermal fibroblasts
on electrospun polycaprolactone collagen nanofibrous membrane. Artif Organs 30:440
28 1 Introduction

160. Sun T, Mai SM, Norton D, Haycock JW, Ryan AJ, MacNeil S (2005) Self-organization of
skin cells in three-dimensional electrospun polystyrene scaffolds. Tissue Eng 11:1023
161. Khil M-S, Cha D-I, Kim H-Y, Kim I-S, Bhattarai N (2003) Electrospun nanofibrous
polyurethane membrane as wound dressing. J Biomed Mater Res Part B Appl Biomater
67B:675
162. Chew SY, Mi R, Hoke A, Leong KW (2009) The effect of the alignment of electrospun
fibrous scaffolds on Schwann cell maturation. Biomaterials 29:653
163. Chew SY, Mi R, Hoke A, Leong KW (2007) Aligned protein-polymer composite fibers
enhance nerve regeneration: a potential tissue-engineering platform. Adv Funct Mater
17:1288
164. Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P, Mey J (2007) Guidance
of glial cell migration and axonal growth on electrospun nanofibers of poly-e-caprolactone
and a collagen/poly-e-caprolactone blend. Biomaterials 28:3012
165. Yang F, Murugan R, Wang S, Ramakrishna S (2005) Electrospinning of nano/micro scale
poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering.
Biomaterials 26:2603
166. Koh HS, Yong T, Chan CK, Ramakrishna S (2008) Enhancement of neurite outgrowth
using nano-structured scaffolds coupled with laminin. Biomaterials 29:3574
167. Bini TB, Wang S, Gao S, Ramakrishna S (2006) Poly(l-lactide-co-glycolide) biodegradable
microfibers and electrospun nanofibers for nerve tissue engineering: an in vitro study.
J Mater Sci 41:6453
168. Yoshimoto H, Shin YM, Terai H, Vacanti JP (2003) A biodegradable nanofiber scaffold by
electrospinning and its potential for bone tissue engineering. Biomaterials 24:2077
169. Erisken C, Kalyon DM, Wang H (2008) Functionally graded electrospun polycaprolactone
and b-tricalcium phosphate nanocomposites for tissue engineering applications.
Biomaterials 29:4065
170. Fujihara K, Kotaki M, Ramakrishna S (2005) Guided bone regeneration membrane made of
polycaprolactone/calcium carbonate composite nano-fibers. Biomaterials 26:4139
171. Sui G, Yang X, Mei F, Hu X, Chen G, Deng X, Ryu S (2007) Poly-L-lactic acid/
hydroxyapatite hybrid membrane for bone tissue regeneration. J Biomed Mater Res 82A:445
172. Kim H-W, Song J-H, Kim H-E (2005) Nanofiber generation of gelatin-hydroxyapatite
biomimetics for guided tissue regeneration. Adv Funct Mater 15:1988
173. Lee SJ, Yoo JJ, Lim GJ, Atala A, Stitzel J (2007) In vitro evaluation of electrospun
nanofiber scaffolds for vascular graft application. J Biomed Mater Res 83:999
174. Stitzel J, Pawlowski KJ, Wnek GE, Simpson DG, Bowlin GL (2001) Arterial smooth
muscle cell proliferation on a novel biomimicking, biodegradable vascular graft scaffold.
J Biomater Appl 16:22
175. Stitzel J, Lee SJ, Komura M, Berry J, Soker S, Lim G, Dyke MV, Czerw R, Yoo JJ, Atala A
(2006) Controlled fabrication of a biological vascular substitute. Biomaterials 27:1088
176. Inoguchi H, Kwon IK, Inoue E, Takamizawa K, Maehara Y, Matsuda T (2006) Mechanical
responses of a compliant electrospun poly(L-lactide-co-e-caprolactone) small-diameter
vascular graft. Biomaterials 27:1470
177. Boland ED, Matthews JA, Pawlowski KJ, Simpson DG, Wnek GE, Bowlin GL (2004)
Electrospinning collagen and elastin: preliminary vascular tissue engineering. Front Biosci
9:1422
178. Vaz CM, van Tuijl S, Bouten CVC, Baaijens FPT (2005) Design of scaffolds for blood
vessel tissue engineering using a multi-layering electrospinning technique. Acta Biomater
1:575
179. In Jeong S, Kim SY, Cho SK, Chong MS, Kim KS, Kim H, Lee SB, Lee YM (2007) Tissue-
engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile
perfusion bioreactors. Biomaterials 28:1115
References 29

180. Soffer L, Wang X, Zhang X, Kluge J, Dorfmann L, Kaplan DL, Leisk G (2009) Silk-based
electrospun tubular scaffolds for tissue-engineered vascular grafts. J Biomater Sci Polym Ed
19:653
181. Shin M, Ishii O, Sueda T, Vacanti JP (2004) Contractile cardiac grafts using a novel
nanofibrous mesh. Biomaterials 25:3717
182. Rockwood DN, Akins RE, Parrag IC, Woodhouse KA, Rabolt JF (2008) Culture on
electrospun polyurethane scaffolds decreases atrial natriuretic peptide expression by
cardiomyocytes in vitro. Biomaterials 29:4783
183. Zong X, Bien H, Chung CY, Yin L, Fang D, Hsiao BS, Chu B, Entcheva E (2005)
Electrospun fine-textured scaffolds for heart tissue constructs. Biomaterials 26:5330
184. Li WJ, Tuli R, Okafor C, Derfoul A, Danielson KG, Hall DJ, Tuan RS (2005) A three-
dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal
stem cells. Biomaterials 26:599
185. Thorvaldsson A, Stenhamre H, Gatenholm P, Walkenström P (2008) Electrospinning of
highly porous scaffolds for cartilage regeneration. Biomacromolecules 9:1044
186. Sill TJ, von Recum HA (2008) Electrospinning: applications in drug delivery and tissue
engineering. Biomaterials 29:1989–2006
187. Agarwal S, Wendorff JH, Greiner A (2008) Use of electrospinning technique for biomedical
applications. Polymer 49:5603–5621
188. Zeng J, Yang L, Liang Q, Zhang X, Guan H, Xu X, Chen X, Jing X (2005) Influence of the
drug compatibility with polymer solution on the release kinetics of electrospun fiber
formulation. J Controlled Release 105:43
189. Xie J, Wang C-H (2006) Electrospun micro- and nanofibers for sustained delivery of
paclitaxel to treat C6 glioma in vitro. Pharm Res 23:1817
190. Huang Z-M, He CL, Yang A, Zhang Y, Han X-J, Yin J (2006) Encapsulating drugs in
biodegradable ultrafine fibers through co-axial electrospinning. J Biomed Mater Res Part A
77A:169
191. Cui W, Li X, Yu G, Zhou S, Weng J (2006) Investigation of drug release and
matrix degradation of electrospun poly(D,L-lactide) fibers with paracetanol inoculation.
Biomacromolecules 7:1623
192. Zeng J, Xu X, Chen X, Liang Q, Bian X, Yang L, Jing X (2003) Biodegradable electrospun
fibers for drug delivery. J Controlled Release 92:227
193. Qi H, Hu P, Xu J, Wang A (2006) Encapsulation of drug reservoirs in fibers by emulsion
electrospinning: morphology characterization and preliminary release assessment.
Biomacromolecules 7:2327
194. Luong-Van E, Grondahl L, Chua KN, Leong KW, Nurcombe V, Cool SM (2006) Controlled
release of heparin from poly(e-caprolactone) electrospun fibers. Biomaterials 27:2042
195. Zhang Y, Wang X, Feng Y, Li J, Lim CT, Ramakrishna S (2006) Coaxial electrospinning
of (fluorescein isothiocyanate-conjugated bovine serum albumin)-encapsulated poly(e-
caprolactone) nanofibers for sustained release. Biomacromolecules 7:1049
196. Casper CL, Yamaguchi N, Kiick KL, Rabolt JF (2005) Functionalizing electrospun fibers
with biologically relevant macromolecules. Biomacromolecules 6:1998
197. Zeng J, Aigner A, Czubayko F, Kissel T, Wendorff JH, Greiner A (2005) Poly(vinyl
alcohol) nanofibers by electrospinning as a protein delivery system and the retardation of
enzyme release by additional polymer coatings. Biomacromolecules 6:1484
198. Richardson TP, Peters MC, Ennett AB, Mooney DJ (2001) Polymeric system for dual
growth factor delivery. Nat Biotechnol 19:1029
199. Chew SY, Wen J, Yim EKF, Leong KW (2005) Sustained release of proteins from
electrospun biodegradable fibers. Biomacromolecules 6:2017
200. Lu YK, Kim K, Hsiao BS, Chu B, Hadjiargyrou M (2003) Development of a nanostructured
DNA delivery scaffold via electrospinning of PLGA and PLA-PEG block copolymers.
J Controlled Release 89:341
30 1 Introduction

201. Kretlow JD, Mikos AG (2008) From material to tissue: biomaterial development, scaffold
fabrication, and tissue engineering. AlChE J 54:3048
202. Chen RR, Mooney DJ (2003) Polymeric growth factor delivery strategies for tissue
engineering. Pharm Res 20:1103
203. Saltzman WM, Olbricht WL (2002) Building dug delivery into tissue engineering. Nat Rev
Drug Discov 1:177
204. Hersel U, Dahmen C, Kessler H (2003) RGD modified polymers: biomaterials for
stimulated cell adhesion and beyond. Biomaterials 24:4385
Chapter 2
Materials and Methods

2.1 Materials

Table 2.1 lists the polyesters used to fabricate scaffolds, specifying compositions,
suppliers and polymer molecular weight distributions. It is pointed out that all
copolymers employed possess a random distribution of their comonomers.
P(L)LA, PLA75GA25, P(LA-TMC), PCL and PEO were commercial polymers
and they were used without further purifications.
P(D,L)LA and PLAGA copolymers, provided by the Institute of Polymers and
Carbon Materials (Zabrze, Poland), were synthesized by ring opening polymeri-
zation using a zirconium-based initiator as previously described [1]. The use of
these low toxicity initiators is particularly interested, as it was demonstrated that
cell viability is higher when polymers are synthesized with zirconium compounds
as catalysts instead of the widely used tin compounds [2]. These polymers were
used after drying at 80 °C under vacuum in order to eliminate residual solvents
employed during polymer purification steps.
Polymers supplied by the Centre for Biocatalysis and Bioprocessing of
Macromolecules (New York, USA) (e.g. PPDL, P(PDL-CL) and P(PDL-DO)) were
synthesized by ring opening polymerization catalyzed by Candida antartica Lipase B
(CALB) as earlier described [3–5]. Enzyme catalyzed polymerizations allow to
synthesize copolymers displaying a random distribution of monomeric units thanks
to transesterification reactions promoted by CALB during the synthesis.
Chloroform (CLF), Dichloromethane (DCM), N,N-dimethylformamide (DMF),
Dimethyl sulfoxide (DMSO), Methanol (MetOH), Tetrahydrofuran (THF),
2-Chloroethanol (CE), Acetone, 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and
Ethanol (EtOH) were purchased by Sigma–Aldrich Co. and they were used without
further purification.
The cationic macroinitiator for the Atom Transfer Radical Polymerization,
Poly(2-(N,N,N-trimethylammonium iodide) ethyl methacrylate-co-bis-2,3-(2-bro-
moisobutyl) glycerol monomethacrylate) (MI; Mn = 21.2 kg/mol, PDI = 1.34, by

C. Gualandi, Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering, 31

Springer Theses, DOI: 10.1007/978-3-642-19272-2_2,
Ó Springer-Verlag Berlin Heidelberg 2011
32 2 Materials and Methods

Table 2.1 Polymers used for scaffold fabrication

Polymer Composition Supplier Molecular weight
(molar ratio) distribution
Poly(L)lactide (Lacea – Mitsui fine chemicals Mw = 172 kg/mol
H.100-E) [P(L)LA] (Dusseldorf, Germany) Mw/Mn = 3.2a
Poly(D,L)lactide D:L = 50:50 Institute of polymers and Mw = 155 kg/mol
[P(D,L)LA] carbon materials, Mw/Mn = 2.4a
polish academy of
science (Zabrze,
Poland)
Poly((L)lactide- LA:GA = 90:10 Institute of polymers Mw = 20 kg/mol
co-glycolide) and carbon materials, Mw/Mn = 2.1b
[PLA90GA10] polish academy of
science (Zabrze,
Poland)
Poly((D,L)lactide- LA:GA = 75:25 Boehringer (Ingelheim, Mw = 170 kg/mol
co-glycolide) Germany) Mw/Mn = 2.0a
(Resomer RG 756 S)
[PLA75GA25]
Poly((D,L)lactide- LA:GA = 65:35 Institute of polymers Mw = 41 kg/mol
co-glycolide) and carbon materials, Mw/Mn = 2.1a
[PLA65GA35] polish academy of
science (Zabrze,
Poland)
Poly((D,L)lactide- LA:GA = 50:50 Institute of polymers Mw = 81 kg/mol
co-glycolide) and carbon materials, Mw/Mn = 2.4a
[PLA50GA50] polish academy of
science (Zabrze,
Poland)
Poly((L)lactide- LA:TMC = 70:30c Boehringer (Ingelheim, d

co-trimethylene Germany)
carbonate) (Resomer
LT 706) [P(LA-TMC)]
Poly(e-caprolactone) (787 – Union carbide Co. (New Mw = 74 kg/mol
Tone) [PCL] Jersey, USA) Mw/Mn = 2.3a
Poly(x-pentadecalactone) – Centre for biocatalysis Mw = 128 kg/mol
[PPDL] and bioprocessing Mw/Mn = 2.0a
of macromolecules,
polytechnic university
(New York, USA)
Poly(x-pentadecalactone- PDL:CL = 69:31 Centre for biocatalysis Mw = 240 kg/mol
co-e-caprolactone) and bioprocessing of Mw/Mn = 8e
[P(PDL-CL)] macromolecules,
polytechnic university
(New York, USA)
Poly(x-pentadecalactone- PDL:DO = 47:53 Centre for biocatalysis Mw = 70 kg/mol
co-p-dioxanone) and bioprocessing of Mw/Mn = 2.3e
[P(PDL-DO)] macromolecules,
polytechnic university
(New York, USA)
(continued)
2.1 Materials 33

Table 2.1 (continued)

Polymer Composition Supplier Molecular weight
(molar ratio) distribution
Poly(ethylene oxide) – Sigma–Aldrich Mw * 1000 kg/
[PEO] (Milan, Italy) molf
a
measured by gel permeation chromatography (GPC) in CLF at 25 °C by using polystyrene
standards
b
measured by GPC in THF at 25 °C by using polystyrene standards
c
mass ratio
d
supplier provides inherent viscosity = 1.4 ± 0.2 dl/g, measured in CLF 0.1% w/V at 25 °C
e
measured by GPC in ortho-dichlorobenzene at 135 °C by using polystyrene standards
f
provided by the supplier

GPC in DMF at 70 °C, by using polymethylmetacrilate standards), was synthe-

sized as previously described [6]. Glycerol Monomethacrylate (GMMA; Cognis,
Southampton, UK), 2,2’-Bipyridine (2,20 -Bpy; Sigma–Aldrich), CuCl and CuBr2
(Sigma–Aldrich) were used without further purification.
A six-arms star-branched oligo(D,L)lactic acid (PLA-T6; Mn = 25 kg/mol by
1
H-NMR), synthesized as described by Biela et al. [7], was kindly provided by Prof.
G. Di Silvestro (Organic and Industrial Chemistry Dept., University of Milan). In
brief, the PLA-T6 was obtained by polycondensation reaction of lactide and the
exa-functional initiator di-pentaerythritol (T6) catalyzed by Sn(Oct)2. In order to
transform the carboxyl end groups of PLA-T6 into carboxilate terminal groups, the
oligomer was subject to the following salification procedure prior to use: 400 mg of
PLA-T6 were dissolved in 20 ml of THF with the addition of 0.6 ml NaOH 0.1 M.
The solution was stirred for 2 h then oligomer was precipitated in cyclohexane,
washed with deionized water and dried over P2O5 under vacuum for 2–3 days.

2.2 Scaffold Fabrication by ScCO2 Foaming

ScCO2 scaffold fabrication was carried out in a 60 mL stainless steel high-pressure

autoclave (made in house) (Fig. 2.1a) connected with a high pressure PM101 pump
(New Ways of Analytics, Lörrach, Germany) that was used to charge CO2 into the
autoclave. Temperature and CO2 pressure inside the autoclave were accurately
controlled during the foaming process by using: (1) a CAL 3300 temperature con-
troller (Advanced Industrial Systems, Inc., Luisville, USA) connected to a thermo-
couple inserted into the autoclave (Fig. 2.1b) and (2) a backpressure regulator
(Bronkhorst, the Netherlands) and a pressure transducer (Fig. 2.1c).
A polymer disc (200 ± 5 mg) inserted in a cylindrical Teflon mould (10 mm
diameter and 10 mm height, Fig. 2.1d) was placed in the autoclave and the
foaming process was carried out as follows: the autoclave was heated to
the desired temperature and filled with CO2 at 230 bar (pressurization stage). The
system was maintained at constant temperature and pressure over a given period of
time (soak time). The soak stage was followed by a depressurization stage during
34 2 Materials and Methods

Fig. 2.1 ScCO2 foaming apparatus a autoclave, b thermocouple, c back pressure regulator
system and d Teflon mould with 12 wells for batch scaffold production. This mould was designed
with a detachable base to allow easy removal of scaffolds after fabrication

Fig. 2.2 View cell for real-

time foaming process
observations

which the pressure was decreased to ambient pressure at controlled depressur-

ization rate (dP/dt). During this stage the temperature was either kept constant or
lowered at a controlled cooling rate (dT/dt) down to a selected temperature. At the
end of the process, after a spontaneous cooling to room temperature (RT), the
Teflon mould containing the foamed sample was removed from the autoclave.
Alternatively, the foaming process was also carried out in a 100 ml stainless
steel high-pressure autoclave equipped with two sapphire windows (view cell,
Fig. 2.2) with the aim to visualize the macroscopic changes of sample aspect and
shape during the foaming process. Sapphire windows were located at each end of
the autoclave, one used for back illumination. A polymer disc (200 ± 5 mg) was
placed in a Teflon mould (10 9 10 9 2 mm) inserted in the view cell, and
foaming process was carried out as previously described. A CCD uEye camera
(Firstsight Vision, UK) placed in front of the sapphire window was used to capture
real time images of the sample subjected to the foaming process.
2.3 Scaffold Fabrication by Electrospinning 35

2.3 Scaffold Fabrication by Electrospinning

The electrospinning (ES) apparatus was placed in a glove box (Iteco Eng.,
Ravenna, Italy, 100 9 75 9 100 cm) equipped with a temperature and humidity
control system (Fig. 2.3b). The ES apparatus (made in house) was composed of a
SL 50 p 10/CE/230 high voltage power supplier (Spellman, New York, USA,
Fig. 2.3c), a KDS-200 syringe pump (KDScientific Inc., Massachusetts, USA,
Fig. 2.3d), a glass syringe containing the polymer solution, a stainless-steel blunt-
ended N-P3-G18 needle (Hamilton, Bonaduz, Switzerland, Fig. 2.3e) connected
with the power supply electrode and a grounded collector (Fig. 2.3f). The polymer

Fig. 2.3 a Scheme of the ES process, b glove box containing the ES apparatus composed of:
c high voltage power supply, d syringe pump, e metallic needle and f collector. g representative
picture of ES mat deposited on aluminium plate collector (10 9 10 mm)
36 2 Materials and Methods

solution was dispensed through a Teflon tube to the needle that was vertically
placed on the target.
According to productivity and fibre deposition distribution requirements, col-
lectors of different type and size were used. Alluminium plate collectors were
employed for fabricating non-woven ES mats composed of randomly oriented
fibres. Cylindrical rotating targets of different radius were used to collect ES fibres
with different degree of spatial orientation. Finally, ad hoc developed targets, that
allow to accurately control fibre deposition, were employed in order to fabricate
patterned ES mats. Such collectors and the effect of their composition and
geometry on mat morphology will be described in detail in Chap. 3.
ES polymer mats loaded with additives (i.e. Endothelial Cell Growth Factor
Supplement and PLA-T6 oligomers) were obtained by simply electrospinning
the polymeric solution containing the additional substance at the desired
concentration.
In all cases, after fabrication, ES mats (Fig. 2.3g) were kept under vacuum over
P2O5 at RT overnight in order to eliminate residual solvents.

2.3.1 Surface Modification

P(L)LA ES samples (3 ± 1 mg) containing 10% w/w of PLA-T6 oligomers were

fixed on plastic rings (CellCrownTM12, inner diameter = 15 mm, Scaffdex,
Tampere, Finland, Fig. 2.4) and were immersed in EtOH for 15 min in order to
ensure a fast and complete wetting of the intrinsically hydrophobic scaffold. EtOH
was then replaced by deionized water through repeated rinses.
Each wet mat was placed in 10 ml of 0.1% w/V aqueous solution of the ATRP-
macroinitiator (MI) and left at RT overnight under shaking to allow electrostatic
adsorption to occur. Then, mats were thoroughly rinsed with deionized water and
dried under nitrogen purge.

Fig. 2.4 ES mat fixed on a

CellCrownTM12 plastic ring
(Scaffdex)
2.3 Scaffold Fabrication by Electrospinning 37

The obtained MI-coated mats were inserted in 50 ml Falcon tubes, placed in a

parallel reactor and purged with nitrogen. Deionised water containing a mixture of
GMMA, CuCl, CuBr2 and 2,20 -Bpy (molar ratio = 60:1:0.3:2.8, GMMA con-
centration = 2.1 M) was bubbled with nitrogen for 45 min before addition of the
required volume of MetOH (H2O:MetOH = 1:1, by volume). Aliquots of this
reactive mixture (22 ml) were then transferred to each Falcon tube containing ES
mats to start polymerisation. The Surface-Initiated ATRP (SI-ATRP) of GMMA
on nanofibers was carried out under nitrogen at RT for 16 h. The polymerization
was interrupted by exposing mats to air. All samples were thoroughly washed with
deionised water for one day.

2.3.2 In Vitro Degradation Experiments

Hydrolytic degradation studies were carried out on P(L)LA non-woven ES mats

(25 ± 5 mg). Prior to degradation experiments specimens were dried over P2O5
under vacuum at RT for 2 days and they were weighted to yield the sample initial
weight (m0). Subsequently, samples were pre-wetted in EtOH for 15 min. EtOH
was then replaced by deionized water through repeated rinses. Wet ES samples
were immersed in phosphate buffered solution (0.1 M, pH = 7.4) and incubated in
a shaking bath (SBS30 Stuart Scientific, Surrey, UK) at 37 °C and 50 revs/min.
The buffer solution was periodically changed to keep the pH constant during the
entire time scale of the degradation experiments. After selected exposure times,
samples were recovered, repeatedly washed with deionized water to remove the
buffer salt components and then dried over P2O5 under vacuum for 2 days to
constant weight (mx). The percentage weight remaining m(%) after buffer expo-
sure was calculated according to Eq. 2.1:
m0 mx
m ð% ) ¼ 100   100 ð2:1Þ
m0
Where m0 is sample initial weight and mx is sample weight after x days in
buffer at 37 °C.

2.3.3 Scaffold Preparation for Cell Culture Experiments

Scaffold fixation on plastic rings (Fig. 2.5a, b) was adopted in order not only to
avoid cell dispersion/outflow during cell culture experiments, but also to improve
scaffold handling and to prevent scaffold shrinkage during the subsequent cell
culture steps. Cell culture experiments were performed by inserting the ES
samples, preliminarily mounted on plastic rings, into common TCPS culture wells
(Fig. 2.5c). During the seeding step, cells were confined onto the upper scaffold
surface by the walls of the ring. By this means, cell migration towards the TCPS
well bottom was prevented (Fig. 2.5d).
38 2 Materials and Methods

Fig. 2.5 a ES scaffold fixed on a Tecaflon plastic ring by using medical-grade silicon, b ES
scaffold fixed on a CellCrownTM6 plastic ring (Scaffdex), c common TCPS multiwell culture
plates (different well dimensions are available) and d schematic representation of cell culture
experiments: the ES sample fixed on the plastic ring is inserted into the TCPS well and cells
are seeded on the upper surface of the scaffold

When cell culture experiments were performed in 12-multiwell TCPS plates

(circular wells of 19 mm in diameter), ES scaffolds were fixed on Tecaflon
(PVDF) plastic rings (internal diameter = 17 mm, external diameter = 18 mm)
using silicone (GE Silicones Rubber, RTV 108Q, Fig. 2.5a). When cell culture
experiments were performed in 6-multiwell TCPS plates (circular wells of 32 mm
in diameter), CellCrownTM6 plastic rings (inner diameter = 29 mm, Scaffdex,
Tampere, Finland, Fig. 2.5) were used to fix the ES scaffolds without the need to
use silicon glue.
Before exposure to culture medium, all scaffolds were subjected to a sterili-
zation procedure using EtOH according to the following protocol: under a laminar
flow, the scaffolds were immersed in 85% V/V EtOH for 15 min, followed by 70%
V/V EtOH for 15 min, and then washed 3 times with phosphate buffered saline
2.3 Scaffold Fabrication by Electrospinning 39

(PBS, pH = 7.4) plus 2% Penicillin/Streptomycin (BioWhittaker-Lonza) and

0.2% Amphotericyn B (Sigma). Scaffolds were kept in this solution overnight
under ultraviolet irradiation (TUV 30 W/G30 T8).

2.4 Characterization Methods

2.4.1 Thermogravimetric Analysis

Thermogravimetric analysis (TGA) measurements were carried out using a

TGA2950 thermogravimetric analyzer (TA Instruments, New Castle, Delaware,
USA). Analysis were performed on samples weighing 2–8 mg, from RT to
600 °C, at a heating rate of 10 °C/min, under N2 flow.

2.4.2 Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) measurements were carried out in helium

atmosphere by using a Q100 DSC apparatus (TAInstruments, New Castle, Dela-
ware, USA) equipped with a liquid nitrogen cooling system (LNCS) low-tem-
perature accessory. Samples were placed in aluminum pans and subjected to
heating scans at 20 °C/min from -80 °C to a temperature higher than glass
transition temperature (Tg) for completely amorphous polymers, or higher than
melting temperature (Tm) when semicrystalline polymers were analysed. Either
quench cooling or controlled cooling at 10 °C/min were applied between heating
scans. Tg values were taken at half-height of the glass transition heat capacity step
while crystallization temperatures (Tc) and Tm were taken at the maximum of
exotherm and endotherm peaks respectively. The degree of crystallinity, vc, was
calculated using the following equation:
DHm
vc ¼  100 ð2:2Þ
DH0m
Where DHm is the experimental melting enthalpy obtained from the DSC scan
and DH0m is the melting enthalpy of 100% crystalline polymer.

2.4.3 Scanning Electron Microscopy

Samples were fixed with a conducting bi-adhesive tape on aluminium stubs and they
were sputter coated with gold. Scanning electron microscopy (SEM) observations
were carried out by using a Philips 515 microscope at an accelerating voltage of
15 kV. Images were acquired and analysed with EDAX Genesis software.
40 2 Materials and Methods

2.4.4 Micro X-Ray Computed Tomography

Micro X-ray Computed Tomography (l-CT) images were acquired using a

Skyscan 1174 Scanner (Skyscan, Aartselaar, Belgium). The scanner was set to a
voltage of 50 kV and a current of 800 mA. By keeping constant the threshold
range, the resulting 2D images were elaborated to obtain 3D reconstructions of the
scaffolds, from which porosity and pore size were calculated, and pore intercon-
nectivity was visually estimated.

2.4.5 Stress–Strain Analysis

Mechanical properties of foamed scaffolds were evaluated on 5 mm 9 5 mm 9

3 mm (thickness) specimens. Compression stress–strain measurements were per-
formed with a TA.HDplus Texture Analyzer (Stable Micro Systems Ltd., Surrey,
United Kingdom) at RT and at a cross head speed of 0.01 mm/s (load cell 750 N).
Triplicate measurements were performed and average values (±standard deviation)
are reported.

2.4.6 Wide Angle X-Ray Diffraction

Wide angle X-ray diffraction (WAXS) measurements were carried out at RT with
a X’Pert PRO diffractometer (PANalytical, Almelo, the Netherlands) equipped
with an XCelerator detector. Cu anode was used as X-ray source (K radiation at
k = 0.15406 nm, 40 kV, 40 mA) and 1/4 divergence slit was used to collect data
in the range 2h = 2–60°. After subtracting the diffractogram of an empty sample
holder from the experimental diffraction curve, the amorphous and crystalline
contributions were calculated by fitting method using the WinFit program. The
degree of crystallinity (vc) was evaluated as the ratio of the crystalline peak areas
to the total area under the scattering curve [8].

2.4.7 Gel Permeation Chromatography

Sample molar mass was evaluated by gel permeation chromatography (GPC) in

chloroform (flow rate = 1 ml/min) at 35 °C by using a VE3580 solvent delivery
system (Viscotek Corp., Texas, USA) with a set of two PLgel Mixed-C columns
and a Shodex SE 61 refractive index detector. A volume of 100 lL of sample
solution in chloroform (5% w/V) was injected. Polystyrene standards were used to
generate a calibration curve.
2.4 Characterization Methods 41

2.4.8 f-Potential

Electrokinetic analyses were performed with a SurPASS electrokinetic analyzer

(Anton Paar, Österreich, Austria) equipped with a cylindrical glass cell. ES samples
pre-wetted in EtOH and thoroughly rinsed with deionized water were analysed. The
wet sample was inserted into the cylindrical cell. The f-potential was determined
from the measurement of streaming potential generated by the imposed movement
of an electrolyte solution (KCl 1 9 10-3 M) through the sample. The f-potential,
which is related to the charge density on sample surface, was determined at pH
values in the range 5–9 by performing automatic titration.

References

1. Dobrzynski P, Kasperczyk J, Janeczek H, Bero M (2001) Synthesis of biodegradable

copolymers with the use of low toxic zirconium compounds. 1. Copolymerization of glycolide
with L-lactide initiated by Zr(Acac)4. Macromolecules 34:5090
2. Czajkowska B, Dobrzynski P, Bero M (2005) Interaction of cells with L-lactide/glycolide
copolymers synthesized with the use of tin or zirconium compounds. J Biomed Mater Res Part
A 74A:591
3. Bisht KS, Henderson LA, Gross RA (1997) Enzyme-catalyzed ting-opening polymerization of
x-pentadecalactone. Macromolecules 30:2705
4. Ceccorulli G, Scandola M, Kumar A, Kalra B, Gross RA (2005) Cocrystallization of random
copolymers of x-pentadecalactone and e-caprolactone synthesized by lipase catalysis. Biomac-
romolecules 6:902
5. Jiang Z, Azim H, Gross RA, Focarete ML, Scandola M (2007) Lipase-catalyzed copolymer-
ization of x-pentadecalactone with p-dioxanone and characterization of copolymer thermal
and crystalline properties. Biomacromolecules 8:2262
6. Edmondson S, Vo CD, Armes SP, Unali GF (2007) Surface polymerization from planar
surfaces by atom transfer radical polymerization using polyelectrolytic macroinitiators.
Macromolecules 40:5271
7. Biela T, Duda A, Penczek S, Rode K, Pasch H (2002) Well-defined star polylactides and thier
behaviour in two-dimensional chromatography. J Polym Sci Part A Polym Chem 40:2884
8. Kakudo M, Kasai N (1972) X-ray diffraction by polymers. American Elsevier Publishing,
New York
Chapter 3
Results and Discussion

The present chapter is divided into four main sections:

1. Porous scaffold fabrication by supercritical carbon dioxide (scCO2) foaming;
2. Porous scaffold fabrication by electrospinning (ES);
3. In vitro hydrolytic degradation of ES scaffolds;
4. Cell culture experiments.
The first two sections illustrate scaffold fabrication technologies employed in
the present research (i.e. scCO2 foaming and electrospinning) and describe porous
scaffold production and characterization. Experiments of scCO2 foaming were
carried out at the Inorganic and Material Chemistry Department and at the Centre
of Biomolecular Science (University of Nottingham). ES technology was imple-
mented in the course of the research thanks to the collaboration with Mechanical
Engineering and Electrical Engineering Departments (University of Bologna).
Finally, the preliminary experiments of ES scaffold surface functionalization,
illustrated at the end of the second section, were carried out at the Laboratory of
Polymers and Biomaterials (University of Manchester).
The third section reports hydrolytic degradation experiments on poly(L)lactide ES
scaffold whereas the fourth section illustrates cell culture experiments performed by
using fibrous scaffolds. The latter were carried out with the collaboration of the
Biochemistry Department ‘‘G. Moruzzi’’ (University of Bologna), the Clinical
Department of Radiological and Histocytopathological Sciences (University of
Bologna) and the Foundation for Development of Cardiac Surgery (Poland).
The two techniques employed to fabricate scaffolds (i.e. scCO2 foaming
and ES) are based on different approaches. The first one uses CO2 as porogen that is
subsequently removed from the polymer matrix, leaving behind a porous structure.
The second one produces a fibre that, thanks to its continuous deposition, generates a
non-woven porous structure. The obtained scaffolds are completely different in terms
of macroscopic aspect, of 3D microscopic morphology and of mechanical properties.
Figure 3.1 shows pictures of both a foamed scaffold fabricated by scCO2 foaming

C. Gualandi, Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering, 43

Springer Theses, DOI: 10.1007/978-3-642-19272-2_3,
 Springer-Verlag Berlin Heidelberg 2011
44 3 Results and Discussion

Fig. 3.1 Pictures and SEM

micrographs of
representatives a foamed
scaffold and b ES scaffold

and of a non-woven mat produced by ES (Fig. 3.1a, b, respectively). The foam is a 3D

object that assumes the shape of the container where it is produced (a cylindrical
mould in this case), whereas the ES scaffold is a flexible sheet less then 1 mm thick.
SEM micrographs show the microscopic morphology of foamed scaffold compared
with that of ES mat: the first one is characterized by large circular pores of hundreds
of microns whereas the second one has much smaller pores, whose dimension is
strictly related to fibre diameter. As a matter of fact, sub-micrometric fibres generate
pores of few microns whereas micrometric fibres lead to pores in the range
10–100 lm [1].
Since mechanical performance is strictly related to the microstructure, the two
kinds of scaffold can be employed for different applications. Foamed scaffolds can
assume a structural supporting role when used as tissue replacements and they are,
therefore, particularly suitable in hard TE (e.g. bone or cartilage TE, depending on
the type of material). On the contrary, ES mats are flexible sheets that deform
easily under bending deformations, being appropriate for replacement of soft tis-
sues such as cardiac, vascular or nervous tissues. Given the intrinsic biomimetic
features of ES materials they are considered the most promising scaffolds in TE.
Therefore, the present research activity was mainly dedicated to study and to
develop ES process and ES products. For the same reason, in the course of the
present Ph.D., cell culture experiments were only carried out on ES scaffolds.

3.1 Porous Scaffold Fabrication by ScCO2 Foaming

Foamed scaffolds have been typically produced to date using amorphous polymers,
in particular non biodegradable polymethylmethacrylates [2–4] and biodegradable
polyesters [5–9]. The classical foaming process exploits the capability of scCO2 to
3.1 Porous Scaffold Fabrication by ScCO2 Foaming 45

plasticize a polymer glass. Typically, polymer foaming (Fig. 3.2) involves three
stages: (1) the pressurization stage (Fig. 3.2 blue), (2) the soak stage (Fig. 3.2 red)
and (3) the depressurization stage (Fig. 3.2 yellow). During the first stage the
glassy polymer absorbs scCO2 that, acting as plasticizer, lowers the Tg and
changes the polymer state from glassy to rubbery. The polymer is maintained at
high constant pressure during the soak stage, where it continues to absorb scCO2.
During the subsequent depressurization stage, run at constant temperature, the
pressure is reduced, CO2 phase changes from supercritical to gas, and bubble
nucleation and gas bubble growth occur in the rubbery polymer, generating pores.
Concomitantly, as a consequence of plasticizer concentration reduction, the
polymer Tg increases and the porous structure is fixed in a solid glassy state
[10–12]. Foam 3D structure strongly depends on process parameters such as
soak time (tsoak), soak pressure (Psoak), depressurization rate (dP/dt) and process
temperature (Tprocess).
ScCO2 foaming is relatively simple to perform for completely amorphous
polymers thanks to the large Tg depression occurring in the presence of scCO2.

Fig. 3.2 Scheme of amorphous polymer foaming process. CO2 pressure changes during the
process (black line) whereas temperature is kept constant (blue line). The dotted blue line depicts
the polymer glass transition temperature changes during the foaming process. Pictures (acquired
with the view cell) show the behaviour of a representative amorphous polyester during the
process: a initial granular polymer, b polymer-scCO2 mixture in the soak stage and c porous
scaffold at the end of the depressurization stage
46 3 Results and Discussion

On the contrary, the foaming process of semicrystalline polymers can be more

complicated, owing to the very low CO2 diffusivity in the polymer crystal phase
[13, 14].
In the course of the present Ph.D., the above described foaming process was
modified and adapted in order to foam a poly(x-pentadecalactone-co-e-caprolac-
tone) (P(PDL-CL), CL = 31 mol%) [15]. This polymer is highly crystalline
(crystallinity degree vc [ 70% by WAXS [16]) with Tm = 82 C (by DSC). The
peculiar crystallization behaviour of P(PDL-CL) copolymers has been recently
investigated [16]. It was demonstrated that, as a result of cocrystallization of the
PDL and CL units, these random copolymers possess a high degree of crystallinity
over the whole composition range, with melting temperature that smoothly
decreases with increasing CL content from that of PPDL (Tm = 100 C) to that of
PCL (Tm = 60 C). Another interesting aspect is that copolymerization of the long
hydrophobic PDL unit with the more hydrophilic CL monomer is expected to
allow tuning of the hydrolytic degradation rate of these materials in view of their
use as bioresorbable scaffolds.
Micro X-Ray Computer Tomography (l-CT) was used to characterize foamed
scaffold morphology. This non-destructive technique employs X-ray to obtain
information on the interior part of the scaffold without sectioning it. During the
scanning, X-rays are attenuated, depending on material density, and they are
captured by the detector. Algorithms calculate attenuation coefficients and the
sample is virtually reproduced after splitting into a series of 2D slices. Image
quality depends on pixel resolution (in the instrument used 1 pixel = 6 lm).
A modelling program stacks the 2D maps to create 3D reconstructions and pro-
vides quantitative information such as scaffold porosity, pore size, wall thickness
and pore interconnectivity.
P(PDL-CL) was firstly subjected to an isothermal foaming process at a tem-
perature lower than Tm = 82 C, to ascertain whether it was possible to foam this
semicrystalline copolymer isothermally (Fig. 3.3). By comparing the images of
P(PDL-CL) sample acquired by means of the view cell, it is clear that no foaming
occurs when the process is carried out at a temperature below the copolymer Tm,
because the final material (Fig. 3.3c) practically maintained the size and shape it
had before the experiment (Fig. 3.3a).
Similar experiments at T \ Tm were reported by Duroidiani et al. [17] who
investigated the foaming of several semicrystalline polymers with different degree
of crystallinity. Their results showed that pores were not spatially homogeneously
distributed in the samples. The authors hypothesized that scCO2 does not penetrate
in the crystal phase and, therefore, pores grow only in the amorphous domains.
Moreover, they found that for highly crystalline polymers no foaming at all
occurred at T \ Tm. Therefore, it is reasonable to conclude that, in the present
case, given the low CO2 diffusivity in the crystal phase, at T \ Tm, there is a large
amount of a solid non-plasticized phase that prevents bubble growth and pore
formation in P(PDL-CL). This suggests that foaming process should be performed
at a temperature higher than P(PDL-CL) Tm, in order to destroy the crystal phase
3.1 Porous Scaffold Fabrication by ScCO2 Foaming 47

Fig. 3.3 Scheme of amorphous polymer foaming process applied to semicrystalline P(PDL-CL).
CO2 pressure changed during the process (black line) whereas temperature was kept constant
(blue line). Pictures (acquired with the view cell) show that P(PDL-CL) sample did not foam
during the process: a starting disc, b polymer-scCO2 mixture and c not foamed P(PDL-CL) disc
at the end of the depressurization stage

and to guarantee a homogeneous absorption of scCO2 by the molten polymer

during the soak stage.
Figure 3.4a shows a picture of P(PDL-CL) obtained after an isothermal
foaming at T [ Tm together with a 2D l-CT sample cross section perpendicular to
the direction of foaming (Fig. 3.4b). It is clear that, when the process was per-
formed at T [ Tm, the sample underwent an increase in height, demonstrating that
a foam formed. No increase in sample diameter occurred, since polymer foaming
was confined in the radial direction by the walls of the mould. These experiments
prove that CO2 can be absorbed by the melt phase during the soak stage. However,
the foamed sample had a hollow in the middle and a moderate number of very
large pores (see Fig. 3.4b), as a consequence of structure collapse at a temperature
that does not allow polymer solidification.
Hence, the foaming procedure was modified as follows. The experiments were
carried out at a soak temperature, Tsoak [ Tm, that was allowed to decrease during
the depressurization stage to a value lower than polymer Tm in order to promote
crystallization and to fix the pores in a solid structure. This alternative foaming
process (Fig. 3.5) applied to the highly crystalline P(PDL-CL) enables to obtain
3D porous foamed scaffolds from this polymer.
48 3 Results and Discussion

Fig. 3.4 Isothermal foaming at T = 90 C (Tm = 82 C, by DCS). Insert: starting P(PDL-CL)
disc, a foamed sample b 2D l-CT of a sample cross section perpendicular to the direction of
foaming (Psoak = 230 bar, tsoak = 20 min, dP/dt = 8 bar/min). Scale bars = 2 mm (Reprinted
from [15], Copyright (2010), with permission from Elsevier)

Fig. 3.5 Scheme of semicrystalline polymer foaming process. Both CO2 pressure (black line)
and temperature (blue line) change during the process. Pictures (acquired with the view cell)
show changes of P(PDL-CL) sample shape during the process: a starting disc, b polymer-scCO2
mixture and c porous scaffold at the end of the depressurization stage

An accurate control of foaming process parameters is necessary in order to

fine tune scaffold 3D structure (e.g. porosity, pore size distribution and pore in-
terconnectivity). Indeed, each parameter has a specific effect on final morphology.
For instance, Tsoak, Psoak and tsoak determine the amount of scCO2 absorbed by the
3.1 Porous Scaffold Fabrication by ScCO2 Foaming 49

polymer melt during the soak stage. Subsequently, during the depressurization
stage, nucleation and growth of CO2 bubbles occurs. According to nucleation
theory [11], the activation energy that must be overcome in order to create stable
nuclei depends on pressure gradient, concentration gradient and temperature [18].
In the present experiments nucleation was forced to occur as a consequence of the
pressure drop that decreased gas solubility and caused CO2 supersaturation in the
polymer. Nucleation density and bubble growth rate depend on depressurization
rate (dP/dt) and on cooling rate (dT/dt). With the aim of understanding the effect of
each variable on scaffold morphology, foaming experiments on the highly crys-
talline P(PDL-CL) copolymer were carried out by changing one process parameter
at a time.
Figure 3.6 compares pictures of samples obtained by applying four different
cooling rates (dT/dt) after soaking at 90 C. 2D l-CT reconstructions of sample
cross sections perpendicular to the direction of foaming, 3D l-CT reconstructions
and pore size distributions are reported. Porosity (p) and average pore size (d)
values are also given. Figure 3.6a and b show that low cooling rates generate large
pores with a high degree of interconnectivity whereas high cooling rates lead to
narrow distributions of small pores with few interconnections (Fig. 3.6c, d).
These results can be explained by considering that bubble growth, pore wall
breaking and pore fixation are controlled by polymer viscosity, which in turn
depends not only on temperature but also on the amount of CO2 dissolved in the
polymer [28]. The latter is regulated by the depressurization rate (dP/dt) which was
kept constant in the present experiments. Therefore, in the applied conditions, the
viscosity of the system essentially depends on cooling rate (dT/dt). At low cooling
rates viscosity increases very slowly and pores grow uncontrolled because of the
high diffusion of CO2 through the highly mobile polymer chains (see Fig. 3.6a, b).
On the contrary, rapid cooling rates lead to a fast increase of viscosity that inhibits
bubble growth, leading to the formation of small closed pores (see Fig. 3.6d). As
pointed out in Chap. 1, a key issue in the field of TE is the obtainment of scaffolds
with interconnected pores. As regards P(PDL-CL) foamed scaffolds, acceptable
pore interconnectivity was achieved by using intermediate cooling rates that pro-
mote formation of pore connections while preventing structure collapse. When
0.23 C/min was applied a scaffold with interconnected pores with average diam-
eter = 255 lm and 70% porosity (Fig. 3.6c) was fabricated. These experiments
show that a very accurate control of the cooling rate is needed in order to carefully
control the 3D structure. Similar conclusions were earlier reported in a study of
scCO2 foaming of P(L)LA, a much less crystalline polyester than P(PDL-CL) [19].
Figure 3.7 shows the effect of soak time on scaffold morphology. A non-homo-
geneous structure, characterized by a non porous core and a porous shell, was
obtained with a short soak time (1 min, Fig. 3.7a). A more homogeneous scaffold
with an average pore size of 155 lm and porosity of 57% was obtained by increasing
the tsoak to 10 min (Fig. 3.7b). A further increase of tsoak (Fig. 3.7c) led to a decrease
of average pore size down to 110 lm, whereas porosity remained almost unchanged.
An analogous influence of soak time on scaffold structure was earlier reported by
Goel et al. [11] and by Tai et al. [5] in foaming amorphous polymers. Therefore, the
50 3 Results and Discussion

Fig. 3.6 Effect of cooling rate on scaffold morphology. P(PDL-CL) foamed at a dT/
dt = 0.15 C/min, b dT/dt = 0.19 C/min, c dT/dt = 0.23 C/min and d dT/dt = 0.27 C/min
(Tsoak = 90 C, Psoak = 230 bar, tsoak = 20 min, dP/dt = 4 bar/min). Scale bar = 1 mm
(Reprinted from [15], Copyright (2010), with permission from Elsevier)

present results suggest that the tsoak parameter similarly affects the scaffold 3D
structure of both semicrystalline and of amorphous polymers. Indeed, in both cases a
polymer matrix, that is a melt in the case of P(PDL-CL) or a rubber when amorphous
polymers are foamed, homogeneously absorbs scCO2. The longer the soak time,
3.1 Porous Scaffold Fabrication by ScCO2 Foaming 51

Fig. 3.7 Effect of soak time on scaffold morphology. P(PDL-CL) foamed at a tsoak = 1 min,
b tsoak = 10 min and c tsoak = 40 min (Tsoak = 90 C, Psoak = 230 bar, dP/dt = 8 bar/min,
dT/dt = 0.27 C/min). Scale bar = 1 mm (Reprinted from [15], Copyright (2010), with
permission from Elsevier)

the higher is the amount of CO2 absorbed. High concentrations of CO2 dispersed in
the polymer generate a high density of nucleation sites during the depressurization
stage, which lead to the fabrication of scaffolds with small pores. Conversely, larger
pore size can be obtained by shortening the soak time. However the results of Fig. 3.7
show that tsoak cannot decrease indefinitely because non-homogenous 3D structures
are obtained when the soak time is too short to allow the homogeneous diffusion of
CO2 throughout the polymer (see Fig. 3.7a).
After investigating the effect of cooling rate and soak time, the influence of
the depressurization rate (dP/dt) on scaffold morphology was evaluated by keeping
constant all other parameters. It is pointed out that experiments that differed in
depressurization rate had to be run at different cooling rate, in order to operate
over the same temperature range. Figure 3.8 shows that fast venting lead to
52 3 Results and Discussion

Fig. 3.8 Effect of depressurization rate on scaffold morphology. P(PDL-CL) foamed at a dP/
dt = 8 bar/min and dT/dt = 0.54 C/min and b dP/dt = 4 bar/min and dT/dt = 0.27 C/min
(Tsoak = 90 C, Psoak = 230 bar, tsoak = 20 min). Scale bar = 1 mm (Reprinted from [15],
Copyright (2010), with permission from Elsevier)

fabrication of structures with small closed pores and low porosity (Fig. 3.8a)
whereas decreasing the depressurization rate helps increasing average pore size,
porosity and pore interconnections (Fig. 3.8b). Indeed, at lower depressurization
rates pore growth lasts longer, thus generating larger and better interconnected
pores. The present highly crystalline and earlier investigated amorphous polymers
[3, 5] show similar dependence of scaffold morphology on depressurization rate.
Indeed, in both cases, CO2 bubbles grow and expand within a polymer matrix
while it is undergoing solidification. Solidification of amorphous polymers occurs
spontaneously during depressurization, where CO2 evaporates, thus plasticizer
concentration in the polymer decreases with a consequent increase of Tg. Differ-
ently to amorphous polymers, P(PDL-CL) solidifies owing to the decrease of
temperature during the process, which induce polymer crystallization to occur.
The above described experiments have demonstrated that scCO2 foaming is a
versatile technique that can be applied to fabricate porous scaffolds not only from
amorphous polymers but also from polymers possessing high crystallinity degree.
However, working with semicrystalline polymers makes the foaming procedure
difficult to control both because the process is non-isothermal and because
solidification implies not only amorphous phase vitrification but also crystalliza-
tion. Crystallization kinetics depend both on CO2 concentration and temperature.
The plasticizer decreases both Tg [20–22] and Tm [23], with Tg decreasing faster
than Tm as a function of plasticizer concentration. This implies that the polymer
3.1 Porous Scaffold Fabrication by ScCO2 Foaming 53

Fig. 3.9 Representative

DSC first scan of P(PDL-CL)
foam (heating rate =
20 C/min) (Reprinted from
[15], Copyright (2010), with
permission from Elsevier)

crystallization window, i.e. the range between Tg and Tm, shifts towards lower
temperature and changes its breadth.
With the aim to analyze possible differences of scaffold crystallinity induced by
changing foaming conditions, the thermal properties of the foamed copolymer
were investigated by DSC. DSC first scans of the starting copolymer and of
scaffolds differently foamed were compared. Surprisingly, all DSC curves were
identical (see Fig. 3.9 as an example) with a single melting peak at 82 ± 1 C and
an associated melting enthalpy of 105 ± 2 J/g, whereas the glass transition heat
capacity step was not detectable owing to the presence of a high amount of crystal
phase. This result shows that crystal phase development in P(PDL-CL) foams is
not affected by cooling rate, depressurization rate and soak time, within the range
investigated. This observation can be attributed to the peculiar crystallization
behaviour of P(PDL-CL) copolymers, where PDL and CL co-units crystallize in
the same lattice with very high crystallization kinetics, thus developing the same
amount of crystal phase regardless of the foaming process conditions. It is pointed
out that the same behaviour is not expected for slowly crystallizable polymers,
such as P(L)LA, for which foaming conditions can be extremely important in
controlling the development of the crystal phase.
The mechanical properties of P(PDL-CL) scaffolds were characterized by
compression tests. Compression experiments on P(PDL-CL) scaffolds yielded
stress–strain curves (see Fig. 3.10 as an example) typical of porous materials [24],
that can be described as follows: (1) an initial linear elastic region controlled by
pore walls bending, (2) a stress plateau due to pore collapse and (3) a stress
increasing region (densification region) where pores are completely collapsed and
the load is applied to a bulk-like material.
Since the above discussed DSC results showed that the ratio of crystal to
amorphous phase was unaffected by the foaming procedure, differences in
mechanical properties must be attributed to scaffold 3-D structure. Table 3.1 lists
the compressive mechanical test results of scaffolds obtained in different foaming
54 3 Results and Discussion

Fig. 3.10 Representative

stress–strain compression
curve of P(PDL-CL) foam.
Three regions can be
distinguished: (i) a linear
elastic region, (ii) a stress
plateau and (iii) a
densification region

Table 3.1 Mechanical properties (from compression stress–strain curves) of selected scaffolds
Foaming conditions Porositya Average E ry (MPa)
pore sizea (MPa)
Tsoak = 90 C, Psoak = 230 bar, tsoak = 20 min, 51 ± 1 186 ± 13 21 ± 2 1.82 ± 0.07
dP/dt = 4 bar/min, dT/dt = 0.38 C/min
Tsoak = 90 C, Psoak = 230 bar, tsoak = 20 min, 61 ± 5 206 ± 7 12 ± 6 1.4 ± 0.4
dP/dt = 4 bar/min, dT/dt = 0.27 C/bar
Tsoak = 90 C, Psoak = 230 bar, tsoak = 20 min, 72 ± 2 272 ± 50 4 ± 1 0.44 ± 0.06
dP/dt = 4 bar/min, dT/dt = 0.23 C/bar
Reprinted from [15], Copyright (2010), with permission from Elsevier
a
from l-CT analysis

conditions together with intrinsic scaffold parameters (porosity and pore size). Both
compressive modulus (E) and compressive strength at yield (ry) decrease with
increasing porosity and pore dimension. In particular, compressive modulus values
are in the same range as that of articular cartilage [25, 26]. Moreover, Fig. 3.10
shows that P(PDL-CL) foams display a linear elastic deformation that extended up
to 10% strain. This preliminary study of scaffold mechanical properties suggests
that P(PDL-CL) foams may find applications as cartilage substitutes.

3.2 Porous Scaffold Fabrication by Electrospinning

By means of a very simple and cost-effective experimental apparatus, ES tech-

nology enables the production of polymeric nanofibres by establishing a high
voltage difference between a metallic needle ejecting the polymeric solution and a
collector. The technique involves an extremely complex electro-fluidodynamical
process that has been the subject of intensive research aimed at modelling the
physics of electrically driven jets and the evolution of jet instability [27–31].
A brief and simplified description of the process can be provided referring to
3.2 Porous Scaffold Fabrication by Electrospinning 55

Reneker’s separation of the ES process into four key stages: (1) launching of the
jet, (2) elongation of the jet straight segment, (3) development of whipping
instability and (4) fibre solidification and collection [32] (Fig. 3.11). In the absence
of an applied electrical field, the droplet of solution coming out from the needle
falls under the influence of gravity. When the solution is positively charged, the
droplet changes its shape from spherical to elliptical. Once a certain threshold
voltage is reached, the drop assumes a conical shape, generating the so called
Taylor’s cone—a term intending to honour Taylor’s contribution to the under-
standing of droplet behaviour in electrical fields [33]—from which a jet emerges.
Jet formation occurs when electrostatic force overcomes the surface tension and
tends to deform the droplet in order to increase the surface area and minimize
charge repulsion (Fig. 3.11i). The same interplay between surface tension and
electrostatic force induces a straight elongation of the jet in the direction of the
collector. The presence of entanglements that physically link the macromolecular
chains allows the formation of a continuous jet (Fig. 3.11ii). During its travel
towards the collector, the jet becomes unstable and displays bending and whipping
movements. Its rapid and symmetric motion generates a cone-shaped envelope. At
this stage the jet reduces its diameter and its surface area increases dramatically
(Fig. 3.11iii). At the end of this stage all the solvent has evaporated and a con-
tinuous single fibre lays randomly oriented onto the collector (Fig. 3.11iv).
Morphology of the collected fibres depends on a number of variables, that are
commonly classified into three groups:
A. solution parameters (e.g. concentration, electrical conductivity, dielectric
constant, viscosity and vapour pressure of the polymer solution, etc.);
B. instrumental parameters (e.g. applied voltage (DV), needle to collector distance
(d), solution flow rate (R), etc.);
C. environmental parameters (e.g. temperature (T), relative humidity (RH), etc.).

Fig. 3.11 Scheme of key

stages of ES process
according to Reneker et al.
[32]
56 3 Results and Discussion

None of the above mentioned variables plays an independent role on the final
fibre morphology. Indeed, a given parameter often differently affects fibre mor-
phology, depending on the specific polymer–solvent combination and on process
variables. For instance, the effect of electrical potential difference DV on fibre
diameter depends on the ES systems considered: DV can either have no effect on
fibre diameter [34, 35], or its increase can decrease fibre diameter [36, 37] or it can
change diameter distribution width [38, 39]. The understanding of how ES vari-
ables interplay in affecting fibre morphology is far from being understood and no
predictive models have been developed. It turns out that suitable ES process
conditions for the obtainment of the desired fibre morphology are always found by
means of a ‘‘trial and error’’ procedure that must be carried out for every polymer.
Despite the fact that the influence of each variable on fibre morphology is often
ambiguous, a number of literature studies have identified few general rules that can
be reasonably applied to all ES systems. It is well-established that solution
parameters, which rely on polymer and solvent properties, are the most important
ones in affecting fibre morphology. Firstly, polymer molecular weight and solution
concentration define polymer processability into fibrous meshes. Indeed, these two
parameters determine the presence of polymeric chain entanglements that provide
a viscoelastic polymer network, essential to prepare ES fibres. For a given poly-
mer, a progressive change from individual electrosprayed beads to continuous
electrospun fibres is usually observed as polymer concentration increases (see
Fig. 3.12 as an example). It turns out that, for a specific polymer solution,
a minimum concentration is required to obtain fibres because, below this value,
viscoleastic forces are not high enough to allow the generation of continuous fibres
and polymer is collected in the form of beads. Some mathematical approaches
have been developed for relating the concentration regime and electrospinnability
of polymer solutions [40, 41].
In addition to polymer concentration and molecular weight, the selected solvent
system profoundly influences fibre morphology [42, 43]. Indeed, electrical prop-
erties and surface tension of the solution interplay in controlling formation and
evolution of the ES jet and they are key factors in determining fibre morphology.
ES is considered a very promising scaffold fabrication technology mainly
because it produces topological biomimetic scaffolds. However, the great interest
in this technology also arises from several other advantages not displayed by other
scaffold fabrication techniques:
• Practically all kind of polymers with high enough molecular weight can be
electrospun into fibres.
• Tuning process parameters enables to regulate fibre morphology in terms of fibre
dimension, presence of beads and fibre surface porosity.
• Fibre deposition and mutual fibre orientation can be easily controlled by acting
on the collector.
• Blended polymer fibres, drug-loaded and particle-loaded fibres can be easily
produced. It is also possible to concomitantly electrospin different fibres to
obtain ‘‘composite’’ non-woven mats.
3.2 Porous Scaffold Fabrication by Electrospinning 57

Fig. 3.12 Representative SEM images of P(D,L)LA in DMF that illustrate fibre morphology
changes as a function of polymer concentration: a at low concentration beads deposit on the
collector, b bead-on string fibres are obtained when concentration is increased and c at higher
concentration bead-free fibres generate

Given the several advantages presented by this technique, the present research
was mainly focused on the implementation of an ES apparatus for the production
of many different ES scaffolds with highly reproducible morphology and on their
application in TE.
A process parameter optimization study is presented in the following section as
an example, whereas next sections describe the main features of the ES scaffolds
produced in the course of the present research project.

3.2.1 Process Conditions Optimization: The Example

of P(PDL-CL)

According to the above discussed considerations, the approach to optimize ES

parameters was always a ‘‘trial-and-error’’ procedure applied to every polymer as
follows. First, given the importance of solution parameters, different polymer
concentrations and several solvent systems were investigated to address their
influence on fibre morphology in terms of viscoelastic and electrical properties and
58 3 Results and Discussion

of solution surface tension. Once a proper polymer solution was selected, instru-
mental parameters were then optimized in order to obtain bead-free sub-
micrometer fibres. The optimization procedure of solution and instrumental
parameters for ES P(PDL-CL) copolymer is discussed as an example (environ-
mental parameters were maintained constant, T = 25 ± 2 C, RH = 40 ± 3%).
Table 3.2 lists physical properties [44] of the most common solvents employed
for ES aliphatic polyesters and their ability to dissolve the copolymer P(PDL-CL).
Among the different solvents tested, CLF is the only pure organic solvent that
dissolves the copolymer at RT. Therefore, solutions of P(PDL-CL), were initially
prepared in CLF. Figure 3.13 shows SEM images of P(PDL-CL) electrospun from
CLF solutions with increasing polymer concentration. At the lowest polymer
concentration (6% w/V, Fig. 3.13a), the polymer collected was in the form of
beaded nanofibres. This type of defect is often eliminated by increasing polymer
concentration which is concomitantly accompanied by increasing of fibre diameter
[40, 45]. Indeed, Fig. 3.13 shows that, by increasing the solution concentration
from 6 to 10% w/V, fibres got thicker, although a high density of elongated beads
still remained (Fig. 3.13b). Additional experiments carried out using a 10% w/V

Table 3.2 Physical properties of selected solvents [44] and their ability to solubilized
P(PDL-CL)
Solvent PPDL Boiling Dielectric constant Surface tension
solubility testa point (C) (dyn/cm) (mN/m2)
Chloroform (CLF) Soluble 61 4.8 26.6
Dichloromethane (DCM) Insoluble 40 8.9 27.2
1,1,3,3,3-Hexafluoro-2- Insoluble 58 16.7 16.1
propanol (HFIP)
2-Chloroethanol Insoluble 129 25.0 38.9
N,N-dimethylformamide Insoluble 153 38.3 35.7
(DMF)
Acetone Insoluble 56 21.0 22.7
Tetrahydrofuran (THF) Insoluble 65 7.5 26.4
Methanol (MetOH) Insoluble 65 33.0 22.1
a
soluble when 3% (w/V) solution is optically clear at room temperature, insoluble when 3%
(w/V) solution is not optically clear at room temperature

Fig. 3.13 SEM micrographs of P(PDL-CL) electrospun from CLF solutions at different polymer
concentrations: a 6% w/V and b 10% w/V (DV = 16 kV, R = 0.01 ml/min, d = 15 cm)
3.2 Porous Scaffold Fabrication by Electrospinning 59

P(PDL-CL) solution in CLF, by changing instrumental parameters (i.e. applied

voltage, needle to collector distance and solution flow rate), did not significantly
improve fibre morphology. Fong et al. [46] hypothesized that beads generate when
surface tension is intermittently higher than electrostatic force and authors stated
that balance of the two opposite effects is predominantly determined by viscosity,
charge density and solution surface tension. Specifically, high viscosity, high
charge density and low surface tension lead to defect-free fibres. In the case of
P(PDL-CL)/CLF solutions, Fig. 3.13 shows that the increase of solution viscosity
did not prevent bead formation. Therefore, alternative mixed solvent systems were
investigated in order to modify electrical properties and surface tension of ES
solutions.
Among the solvents reported in Table 3.2, DMF exhibits the higher dielectric
constant. A small amount of it added to a P(PDL-CL)/CLF solution is expected to
increase the overall dielectric constant of the polymer solution, so that the latter
can carry a higher amount of charge [43] (assuming that solvent properties do not
change dramatically upon addition of P(PDL-CL) [47]). However, even a small
amount of DMF added to a P(PDL-CL)/CLF solution resulted in polymer pre-
cipitation. Hence, the CLF/DMF mixed solvent was discarded.
HFIP possesses surface tension and dielectric constant values that might be a
good compromise between the demand to increase the charge density and to
decrease surface tension of the polymer solution in order to eliminate beads.
Indeed, a jet carrying many charges should act against the tendency of the surface
tension to maintain the bead shape and it is expected to easily elongate. Another
interesting solvent is CE that, despite having a higher surface tension than HFIP,
possesses a higher dielectric constant. Figure 3.14 shows fibres obtained from 6%
w/V P(PDL-CL) in mixed solvent of CLF/HFIP (80/20 by volume, Fig. 3.14b) and
CLF/CE (80/20 by volume, Fig. 3.14c), together with P(PDL-CL) obtained from
6% w/V in CLF for comparison (Fig. 3.14a) (keeping constant all the instrumental
parameters). It is evident that the addition of a co-solvent possessing a high
dielectric constant helps in obtaining less beaded fibres. However, CE has a better
effect compared to HFIP in reducing bead density, probably thanks to its higher
dielectric constant.

Fig. 3.14 SEM micrographs of P(PDL-CL) electrospun from 6% w/V in: a CLF, b CLF/
HFIP = 80/20 and c CLF/CE = 80/20 (DV = 16 kV, R = 0.02 ml/min, d = 15 cm)
60 3 Results and Discussion

The influence of electrical properties on fibre morphology is even more evident

when the amount of CE is increased to the detriment of CLF. Figure 3.15 shows
SEM images of fibres obtained from 6% w/V P(PDL-CL)/CLF solutions con-
taining different amounts of CE. By increasing CE content in the mixed solvent
from 20 to 30% and 40% (Fig. 3.15a–c, respectively), the obtained fibres get
thinner.
A further decrease of fibre dimension can be achieved by decreasing polymer
concentration. Figure 3.16 shows P(PDL-CL) fibres obtained by changing the

Fig. 3.15 SEM micrographs of P(PDL-CL) electrospun from 6% w/V in: a CLF/CE = 80/20,
b CLF/CE = 70/30 and c CLF/CE = 60/40 (DV = 13 kV, R = 0.01 ml/min, d = 15 cm)

Fig. 3.16 SEM micrographs of P(PDL-CL) electrospun from a mixed solution of CLF/CE = 70/
30 (by volume) at: a 8% w/V, b 7% w/V, c 6% wt/V and d 5% w/V (DV = 13 kV, R = 0.01 ml/
min, d = 15 cm)
3.2 Porous Scaffold Fabrication by Electrospinning 61

polymer concentration in CLF/CE (70/30 by volume) from 8% w/V down to 7%,

6% and 5% w/V. The obtained fibres had a diameter distribution of
1080 ± 260 nm, 820 ± 130 nm, 730 ± 180 nm and 500 ± 130 nm respectively.
Although solution parameters are the most important ones in determining
polymer electrospinnability and fibre morphology, the latter is also affected by
instrumental parameters, even if to a limited extend. Figure 3.17 shows P(PDL-
CL) fibres obtained from the same polymer solution but changing the solution flow
rate. The flow rate increase from 0.01 to 0.02 ml/min led to a slight increase of
fibre dimension from 500 ± 130 to 570 ± 170 nm. Commonly, high flow rate
enables the generation of thicker fibres, since it provides a larger amount of
solution to be stretched [45].
Figure 3.18 shows the effect of needle-to-collector distance on P(PDL-CL) fibre
morphology: by increasing needle-to collector distance from 15 to 20 cm, fibres
got thicker (diameter distribution changed from 500 ± 130 to 630 ± 180 nm).
Needle-to collector distance influences both the time of jet elongation and the
electrical field, therefore the jet elongation force. Its effect can be reasonably

Fig. 3.17 SEM micrographs of P(PDL-CL) electrospun from a 5% w/V mixed solution of CLF/
CE = 70/30 at: a R = 0.01 ml/min and b R = 0.02 ml/min (D = 13 kV, d = 15 cm)

Fig. 3.18 SEM micrographs of P(PDL-CL) electrospun from a 5% w/V mixed solution of CLF/
CE = 70/30 at: a d = 15 cm and b 20 cm (DV = 13 kV, R = 0.01 ml/min)
62 3 Results and Discussion

explained by taking into account the two extreme situations: very low distances
generate high electrical fields that stretch the jet for very short times whereas very
high distances generate low electrical fields to stretch fibres over long distances.
In both cases thicker fibres are expected than those obtained at intermediate needle-
to-collector distances where strong enough fields for long enough time are applied
to stretch the fibres. Obviously, the best range of needle-to collector distance strictly
depends on all other ES conditions, in particular on the applied voltage.
Experiments carried out on 5% w/V P(PDL-CL) solution of in CLF/CE = 70/
30 by changing the applied voltage did not provide significantly different fibre
dimensions, so that the applied voltage seemed to have no evident effect on fibre
morphology. Again, it is pointed out that each ES variable can affect fibre mor-
phology depending on the set of conditions used. It can be concluded that the
applied voltage does not have any effect within the range of ES conditions
investigated. However, in general, applied voltage can have a deep influence on
fibre morphology, especially on the occurrence of bead-on-string fibres. Indeed, it
directly controls the amount of charge on solution drop that, overcoming the
surface tension, is responsible to increase the jet surface area [48].
Optimization study of process parameters applied to P(PDL-CL) copolymer led
to identify the following ES conditions for the production of defect-free sub-
micrometric fibres:
• P(PDL-CL) solution: 5% w/V in CLF/CE (70/30 by volume),
• applied voltage: DV = 16 kV,
• solution flow rate: R = 0.01 ml/min,
• needle to collector distance: d = 15 cm.

3.2.2 Electrospun Polymers

ES scaffolds from different degradable polyesters are presented in this section.

In the course of the present research both commercial and non-commercial
polyesters were electrospun into fibres. The commercial polymers used in the
course of this Ph.D. (i.e. polylactides and their copolymers and poly(e-caprolac-
tone)) have been more intensively investigated in the literature as bioresorbable
materials than any other degradable polymer. These materials have been already
successfully used in approved medical devices. Indeed, they are biocompatible and
non-toxic and their use is approved in most of developed countries. Their intensive
use in biomedical research is justified by the fact that bringing to market
implantable scaffolds prepared from approved polymers is quicker and cheaper
than using novel polymers whose biocompatibility is still not attested.
ES conditions parameters were optimized in order to obtain defect-free fibres
by evaluating fibre morphology by Scanning Electron Microscopy. The optimi-
zation study was carried out for every polymer as previously described for P(PDL-
CL). Both pristine polymers and the corresponding ES fibres were characterized by
3.2 Porous Scaffold Fabrication by Electrospinning 63

Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC).

TGA did not reveal residual solvents in the ES fibres described below and con-
firmed that ES process did not affect polymer thermal degradation behaviour. DSC
was used to evaluate the ratio of crystal to amorphous phase in ES fibres. From
DSC first scans emerged that, in some case, this ratio differed from the one of the
pristine material. On the contrary, DSC second scans of pristine polymers and of
the corresponding fibres were always identical.

3.2.2.1 Polylactide

Lactic acid is in many natural products. It is an optically active molecule that

exists both as D or L stereoisomer. Commonly, lactide polymers are synthesized via
ring opening polymerization of the cyclic dimer lactide (either (L,L) or (D,L)-
lactide) and polylactide properties vary depending on the ratio of the two ster-
eoisomers. The optically pure polymers are semicrystalline and, if the other
enantiomer molar content is less than 20%, macromolecules are still able to
crystallize [49].
Since polylactides are relatively unstable in the presence of moisture, their use
was not widespread till 1960, when their benefits in the biomedical field became
evident. Polylactides are biodegradable polymers that offer the great advantage of
maintaining their functionalities for a limited period of time, after which they are
naturally resorbed by the body. Semicrystalline polylactides are mainly used as
load-bearing implants in orthopaedic fixations (e.g. screws, pins) [50] whereas
amorphous polylactides are often employed in drug delivery devices [51] where a
homogeneous dispersion of the active species within a monophasic matrix is
required. With the development of TE principles, polylactide and, more in general,
lactide-based copolymers became perhaps the most commonly used polymers for
scaffold fabrication. Moreover, polylactides are employed for packaging, agri-
cultural and disposable applications. The intrinsic biodegradability and the deri-
vation from renewable resources allow, on one hand, to reduce the social waste
problem and, on the other hand, to reduce the use of carbon fossil-derived
materials.
Amorphous poly(D,L)lactide and semicrystalline poly(L)lactide were both
electrospun by using process conditions listed in Fig. 3.19. The figure also shows
SEM micrographs and fibre diameter distributions of the obtained scaffolds.
Table 3.3 provides calorimetric data obtained from DSC first scans of P(D,L)LA
and P(L)LA samples. Given the absence of backbone stereoregularity, P(D,L)LA is
not expected to develop a crystalline phase during ES process. On the contrary,
P(L)LA may be able to crystallize. P(L)LA starting sample is semicrystalline, with
a melting endotherm peak at Tm = 162 C and a melting enthalpy DHm = 35 J/g,
corresponding to a crystallinity degree vc = 37% (calculated according to Eq. 2.2,
assuming DHm = 93 J/g [52]). DSC first scan of P(L)LA fibres shows a cold
crystallization exotherm peak followed by a melting endotherm of the same entity
(DHc = DHm = 31 J/g). Therefore, P(L)LA fibres reported in Fig. 3.19 appears to
64 3 Results and Discussion

Fig. 3.19 P(D,L)LA and P(L)LA ES scaffolds: process conditions, SEM micrographs and fibre
diameter distributions

Table 3.3 Calorimetric data of P(D,L)LA and P(L)LA samples

Polymer Tg DCp Tc DHc Tm DHm DHm-DHc vcd
(C) (Jg-1C-1) (C) (Jg-1) (C) (Jg-1) (Jg-1)
P(D,L)LA as 54 0.54 – – – – – –
receiveda
ES P(D,L)LAa, c
54 0.39 – – – – – –
P(L)LA as 64 0.15 – – 162 35 35 37
receivedb
ES P(L)LAb, c 61 0.49 86 31 159 31 0 0
a
First scan from -50 to 120 C, heating at 20 C/min
b
First scan from -50 to 200 C, heating at 20 C/min
c
ES mats obtained by using process conditions listed in Fig. 3.19
d
Calculated according to Eq. 2.2, by assuming DHm = 93 J/g [52]

be completely amorphous. P(L)LA is a slow crystallisable polymer compared to

many conventional thermoplastic polymers [53, 54]. During ES process, polymer
chains have little time to organize in a crystal structure before the occurring of
fibre solidification and polymer crystallization can be inhibited if crystallization
rate is low.

3.2.2.2 Poly(lactide-co-glycolide) Copolymers

Properties of polylactide can be adapted to a wide range of possible applications

through copolymerization of lactide with other monomers, the most intensively
3.2 Porous Scaffold Fabrication by Electrospinning 65

investigated one being glycolide. PLAGA copolymers are biomedical commercial

products since 1970 and they have been developed mainly for
resorbable sutures (trade names Vicryl and Polyglactin 910) but they are also
employed in craniomaxillofacial reconstruction and other orthopaedic applications
[50]. The lactide-glycolide molar ratio regulates mechanical properties and phase
morphology but, above all, it affects the rate of degradation. The presence of the
hydrophilic GA units increases the water uptake of the copolymers, thus accel-
erating ester cleavage and giving the possibility to tune resorption rate of the
implant in the organism. Amorphous copolymers with high glycolide content are
commonly resorbed within 1–2 months. When glycolide content is low, resorbtion
times increase up to 6 months, a time still considerably lower than that required for
resorption of the homopolymer polylactide (more than 1 year) [51]. However, the
intensive clinical use of PLA and PLAGA copolymers highlighted that inflam-
matory reactions are the most serious complications associated with these mate-
rials. Inflammations are probably related to the release of acidic products as a
consequence of polymer degradation. This problem might be reduced by using
more hydrophobic polymers that degrade slowly, thus obtaining the release of a
lower amount of acid products.
The copolymers employed in the present research have different lactide-
glycolide molar ratio: PLA90GA10, PLA75GA25, PLA65GA35 and PLA50GA50. They
were all synthesized from (D,L)-lactide apart from the copolymer PLA90GA10 which
was synthesized from the stereoregular (L,L)-lactide. Therefore, PLA75GA25,
PLA65GA35 and PLA50GA50 copolymers re not expected to develop a crystal
phase whereas PLA90GA10 may be able to develop a crystal phase given the
stereoregularity of the lactide units. With the increase of the amount of glycolide
unit, that has higher mobility then lactide unit, the copolymer Tg decreases as
reported in Fig. 3.20 (Tg of polyglycolide taken from [51]).
Figure 3.21 reports optimized process conditions employed in the present
research to electrospin PLA90GA10, PLA75GA25, PLA65GA35 and PLA50GA50.

Fig. 3.20 Glass transition

temperature of PLAGA
copolymers as a function of
glycolide content
66 3 Results and Discussion

Fig. 3.21 PLAGA ES scaffolds: process conditions, SEM micrographs and fibre diameter
distributions

DSC analysis showed that all ES fibres shown in Fig. 3.21 are completely
amorphous.

3.2.2.3 Poly(lactide-co-trimethylene carbonate)

Another interesting copolymer of lactic acid is P(LA-TMC). The addition of TMC

units decreases Tg and confers elastic properties to the materials making P(LA-
TMC) copolymers good candidates for soft tissue reconstruction [55, 56].
3.2 Porous Scaffold Fabrication by Electrospinning 67

P(LA-TMC) employed in the present research is a random copolymer of

(L,L)-lactide with a TMC content of 30% in weight. It has a Tg around 34 C, close
to the physiological temperature. Figure 3.22 shows sub-micrometric P(LA-TMC)
fibres obtained by using optimized process conditions [57].
P(LA-TMC) ES fibres are completely amorphous with a Tg around 34 C (by
DSC first scan, heating at 20 C/min, not shown). Shape memory properties of
P(PLA-TMC) ES mats have been recently observed in a preliminary study carried
out in the laboratory [57]. Since the switching temperature associated with the
shape recover is closed to physiological conditions, these ES scaffolds are
potentially extremely interesting.

3.2.2.4 Poly(e-caprolactone)

Poly(e-caprolactone) is a biocompatible polymer more hydrophobic than both PLA

and PLAGA copolymers, it degrades slower and it is therefore suitable for long-
term applications. In particular a long-term implant called Capronor has been
intensively studied as contraceptive deliverer [58].
Figure 3.23 reports SEM micrographs of micrometric PCL fibres.

Fig. 3.22 P(LA-TMC) ES scaffolds: process conditions, SEM micrograph and fibre diameter
distribution

Fig. 3.23 PCL ES scaffolds: process conditions, SEM micrograph and fibre diameter distribution
68 3 Results and Discussion

Table 3.4 Calorimetric data of PCL samples

Polymer Tg DCp Tc DHc Tm DHm DHm-DHc vcc
(C) (Jg-1C-1) (C) (Jg-1) (C) (Jg-1) (Jg-1)
PCL as -59 0.08 – – 62 88 – 63
receiveda
ES PCLa, b -63 0.10 – – 57 77 – 55
a
First scan from -100 to 130 C, heating at 20 C/min
b
ES mats obtained by using process conditions listed in Fig. 3.23
c
Calculated according to Eq. 2.2, by assuming DHm = 140 J/g [61]

PCL is a semicrystalline polymer that, unlike P(L)LA, is able to crystallize

during ES process. Table 3.4 compares calorimetric data obtained from first DSC
scans of the pristine sample and of ES PCL fibres. The crystallinity degree of ES
fibres appears to be lower than that of the ‘‘as received’’ sample. This result is not
surprising since the fast ES process is expected to limit crystal formation [59, 60]
or even to totally inhibit crystallization, as above reported in the case of P(L)LA.

3.2.2.5 Poly(x-pentadecalactone) and

Poly(x-pentadecalactone-co-p-dioxanone)

PPDL and its copolymers are degradable materials that, despite not being medi-
cally approved yet, display different and extremely interesting properties with
respect to the commonly used approved biomaterials. Firstly, given the presence of
x-pentadecalactone units with 14 methylene units per ester group, they are slowly
degradable polymers. This might reduce inflammatory reactions caused by fast
release of acidic by-products and they might be particularly interesting for long-
term applications. Moreover, PDL has been successfully copolymerized with
comonomers such as trimethylene carbonate [62], e-caprolactone [16], and
p-dioxanone [63], thus enabling material scientists to tailor corresponding
hydrolysis rate and biomaterial physical properties for targeted applications.
In the present research PPDL and a P(PDL-DO) copolymer have been electro-
spun, as well as a P(PDL-CL) copolymer already described in the previous
Sect. 3.2.1. Both PPDL homopolymer and its copolymers used in the present
research are highly crystalline materials. The peculiar crystallization of P(PDL-CL),
where PDL and CL units cocrystallize in the same lattice [16] displaying a iso-
morphic behaviour, has been already discussed in Sect. 3.1. Similarly to P(DL-CL),
in P(DL-DO) the two monomeric units cocrystallize in the same lattice, with a
behaviour, in this case, typical of a isodimorphic system [63]. Figure 3.24 lists
optimized process conditions for obtaining sub-micrometric bead-free PPDL and
P(PDL-DO) fibres together with corresponding SEM micrographs and fibre diameter
distributions. Calorimetric analysis of fibres reported in Fig. 3.24 shows that both
PPDL and P(DL-DO) copolymer are able to crystallize during fibre solidification.
Similarly to PCL, PPDL and P(DL-DO) fibres are less crystalline than the corre-
sponding starting polymers (by DSC analysis).
3.2 Porous Scaffold Fabrication by Electrospinning 69

Fig. 3.24 PPDL and P(PDL-DO) ES scaffolds: process conditions, SEM micrographs and fibre
diameter distributions

3.2.3 Fibre Morphologies

The adjustment of ES parameters allows obtaining many different fibre morphol-

ogies in terms of presence of beads and bead shape, fibre porosity and dimension.
Section 3.2.1 has already introduced the so-called ‘‘bead-on-strings’’ morphology
whose formation has been attributed to surface tension forces intermittently over-
coming electric and the viscoelastic forces during jet elongation [46]. Commonly,
beads have either spherical or ‘‘spindle-like’’ aspects (Fig. 3.25a, b, respectively)
but also more ‘‘exotic’’ shapes can be obtained, such as hollow beads and porous
beads (Fig. 3.25c, d, as examples). Cup-shaped beads and ‘‘prune-like’’ beads have
also been reported in the literature [48, 64]. Commonly, beads are considered
defects along the fibre and process optimization studies usually aim at reducing
their density. Indeed, high bead densities are responsible of worsening mechanical
resistance of fibrous mats, sometimes preventing mat detachment from the collector
without damaging it. However, the presence of beads along the fibres might be
useful for diffusion-controlled drug release application, even if this possibility has
not been explored yet. Indeed, a drug located at the bead core should diffuse out
from the polymer more slowly than a drug located inside the thin fibre.
As already discussed in the previous sections, the control of ES parameters
allows to tailor fibre diameters within a dimensional range from several microns
down to few hundreds of nanometers. As a matter of fact, fibre thickness deter-
mines pore dimension in the fibre mat: non-woven mats with wider interstices can
70 3 Results and Discussion

Fig. 3.25 Representative SEM micrographs of fibres displaying: a spherical beads, b ‘‘spindle-
like’’ beads (red arrows), c hollow beads and d porous beads

be fabricated by increasing fibre diameter [1]. In view of using ES materials as cell

culture supports, pore dimension is particularly important. Indeed, cells can easily
penetrate a 3D structure with pore dimension comparable with that of cells,
whereas the infiltration in smaller pores can be limited.
Fibre diameter also modulates fibre surface area: thinner fibres lead to porous
mats with higher surface to volume ratio. In biomedical applications, surface area
is a key factor since cell-device interactions occur at biomaterial interface.
Moreover, surface area is particularly relevant when scaffolds are loaded with
drugs that are intended to be released in a controlled manner.
Besides fibre diameter, surface area is also determined by fibre surface porosity
and roughness. ES technique allows to produce porous fibres that, as a matter of
fact, display a larger surface area with respect to smooth fibres of similar dimen-
sion. Pore generation at fibre surface can be achieved by appropriately changing ES
conditions, such as solvent system and relative humidity. Figure 3.26a, b show
porous fibres obtained by electrospinning a P(L)LA 20% w/V in DCM and in CLF
respectively. DCM generates thinner fibres than CLF because of its higher
dielectric constant. Interestingly, depending on the solvent, fibres display different
porosity patterns. It has been speculated that pores generate as a consequence of
phase separation during fast solvent evaporation [37]. This hypothesis is supported
by the fact that the addition of a high boiling point solvent—such as DMF—to both
3.2 Porous Scaffold Fabrication by Electrospinning 71

ES solutions inhibits pore formation (not shown). Fibre porosity can be also
controlled by relative humidity: surface pore density can be reduced by decreasing
moisture (see Fig. 3.27). This finding is attributed to the condensation of water
droplets at the surface of the polymer jet, which is undergoing cooling as a

Fig. 3.26 SEM micrographs of porous P(L)LA fibres obtained by electrospinning a solution of
P(L)LA 20% w/V in: a DCM and b CLF

Fig. 3.27 SEM micrographs of P(PDL-DO) fibres obtained by electrospinning a solution of

P(PDL-DO) 30% w/V in DCM/DMSO (80/20 by volume) at: a RH = 55%, b RH = 45%,
c RH = 35% and d RH = 25%. Relative humidity was the only parameter varied during this
experiment
72 3 Results and Discussion

consequence of fast solvent evaporation. The imprints of these droplets, whose

density is higher at high RH values, leave behind a porous surface [37, 65].

3.2.4 Fibre Deposition Geometries

In the most common ES setup fibres are collected on a stationary plane metallic
target as non-woven meshes of randomly oriented fibres. The poor control of
spatial deposition distribution is a direct consequence of the chaotic path of the
charged jet that travels towards the metallic plate. Control of the jet path and of
fibre alignment has been faced by several researchers and it has been summarized
in a comprehensive review by Teo et al. [66], who highlighted the key role of
material and geometry collector in guiding fibre deposition. Fibre alignment is
commonly achieved by using either a high speed rotating tool (e.g. a cylinder [67]
or a sharp edged disk [68]) or auxiliary guiding electrodes [69–71]. Then special
collector geometries have been used to achieve fibre alignment, such as parallel
conducting strips spaced apart by an insulating gap [72, 73] or multiple gold
electrodes deposited on quartz wafers [72, 74]. However, these latter approaches
enable to align fibres only in a confined limited space. Fibre patterning has been
achieved by using conducting grids [75, 76], although the obtainment of complex
patterns still remains a challenge.
The possibility to produce ES scaffolds having controlled fibre deposition
geometries opens new routes towards the investigation of cell-material interactions
and of cell behaviour addressed by substrate morphology.

3.2.4.1 Aligned Fibres

In the course of the present research, highly aligned fibres were produced by using
a metallic cylindrical rotating collector specifically designed for this scope
(Fig. 3.28). A thermoplastic solvent-resistant (POM-H) support that can locate
cylindrical targets of different dimensions (maximum length = 22 cm, maximum
radius = 4 cm) is used. The engine enables cylinder rotation up to a maximum
angular speed x = 6200 rpm.
It is pointed out that rotational speed can be expressed either as angular speed
(x) or as surface linear speed of the collector (v). By keeping constant the angular
speed, the surface linear speed increases with increasing of cylinder radius
(Fig. 3.29).
The relation between angular and linear speed implies that, by keeping constant
the angular speed (x), different linear rates (v) can be produced by simply
changing the cylinder radius (see Fig. 3.30). In this study higher alignment was
achieved by using a larger collector, confirming that surface linear rate of the
cylinder is the parameter that controls the degree of fibre alignment.
3.2 Porous Scaffold Fabrication by Electrospinning 73

Fig. 3.28 Rotating tool for the production of aligned fibres. Cylinders with different radius can
be used

Fig. 3.29 Relation between

angular speed (x) and linear
speed (v) of the cylinder.
Fibres align parallel to the
linear rate, which is
tangential to the collector
surface

Fig. 3.30 SEM micrograph of P(L)LA fibres deposited on mandrel collectors rotating at angular
rate x = 6200 rpm. a mandrel radius = 25 mm, linear rate = 16.2 m/s and b mandrel
radius = 16 mm, linear rate = 10.4 m/s. The arrow indicates the linear rate direction
74 3 Results and Discussion

Fig. 3.31 SEM micrograph of P(L)LA fibres deposited on a cylinder collector (r = 25 mm)
rotating at linear rate: a v = 16.2 m/s, b v = 13.9 m/s and c v = 9.9 m/s. The arrow indicates
the linear rate direction

The movement of the cylinder is responsible of mechanical aligning the fibres

along the rotation direction. Indeed, during fibre formation, the jet travels at a very
high speed and tends to deposit randomly on the collector surface. However, if the
collector moves at a speed higher than that of fibre deposition, fibres can wind
around the cylinder and align along its circumference [77]. Figure 3.31 shows that
fibres, deposited on the same target rotating at different speeds, display a higher
degree of alignment when higher rotational speed is used.

3.2.4.2 Patterned Non-woven Mats

Patterned mats can be produced by acting on material collector and geometry.

In the course of the present research, non-woven scaffolds, displaying patterns on a
different dimensional range, were produced by using two types of target:
• plane collectors made of a conducting metallic substrate coated with a doped
dielectric material, supplied by Smaltiflex SpA (Modena, Italy). Fibres depos-
ited on these collectors generate ‘‘star-shaped’’ patterns, where ‘‘stars’’ of about
50 lm in dimension are spaced out hundreds of microns [78];
• plane collectors made of an array of pins fixed on an insulating material. In this
case, a macroscopic pattern, where local fibre orientation is controlled by the pin
array geometry, is produced.
Collectors supplied by Smaltiflex were made of a metallic substrate coated with
an enamel layer1 [78]. The main enamel components are refractories (quartz,
feldspar, clay) and fluxes (borax, soda ash, cryolite, fluorspar), together with
opacifiers, colours, floating agents and electrolytes [79, 80]. The interest in cou-
pling ES technology with vitreous enamel is related to the specific electrical
properties of enamel. Indeed, enamel is a dielectric material possessing high
electrical resistivity that can be modified by varying the content of the alkaline
oxides, being alkali metal cations the actual charge carriers.

1
Enamel raw materials were prepared by using a home-made frit with the following
composition (in wt%): SiO2: 43, Al2O3:1, B2O3: 13, Na2O: 8, K2O: 5, ZnO: 1, TiO2: 22, P2O5: 3,
F2: 4, that gives a final white colour coating.
3.2 Porous Scaffold Fabrication by Electrospinning 75

Fig. 3.32 Schematic illustration of the electrostatic phenomena occurring during the ES process
using enamelled collectors. a starting situation before fibre deposition and b after deposition of a
layer of polymer fibres (Reprinted from [78], Copyright (2010), with permission from Springer
Science)

In general, the use of a dielectric collector affects the fibre discharging process.
For the sake of clarity, Fig. 3.32 sketches the electrostatic phenomena occurring
when an enamelled steel target is used to collect fibres. When a high voltage
difference is applied between the metallic capillary ejecting the polymeric solution
and the grounded collector, the enamel dielectric layer is subjected to polarization
(Fig. 3.32a). As soon as fibres reach the collector, they tend to rapidly discharge.
However, when a uniform layer of fibres is deposited (Fig. 3.32b), fibre discharging
becomes less efficient and the collector surface, being covered by partially charged
fibres, acquires a positive charge density (r) that decreases with time due to charge
relaxation process across the dielectric coating. The relaxation process is strictly
dependent on enamel electrical properties, in particular on its electrical resisitivity.
The positive residual charge density (r) on collector surface generates a
repulsive force towards the incoming fibres, that is larger at higher collector
resistivity values. The presence of the residual charge r mainly affects fibre
packing density. Since the electrical resistance of the enamelled collectors increase
with enamel coating thickness [78], by using collectors with a chemically identical
enamel layer, but with different thickness,2 it is possible to tailor the fibre packing
density. Figure 3.33 shows that mat section displays a less packed fibre density

2
The enamel raw material was prepared by milling the frit for about 11 h in order to obtain a
powder that was deposited electrostatically over a 0.8 mm thick steel sheet. Enamel coating
thickness was controlled during the deposition process and collectors with different enamel
thickness (0.15 and 0.53 mm) were prepared. The coated specimens were fired in a radiant
furnace with oxidizing atmosphere at 850 C for 6.5 min.
76 3 Results and Discussion

Fig. 3.33 Effect of enamel layer thickness on fibre packing density. SEM micrographs (two
different magnifications) of P(L)LA ES mat sections obtained from collectors with different
enamel thickness: a 0 mm (uncoated steel), b 0.15 mm and c 0.53 mm (Reprinted from [78],
Copyright (2010), with permission from Springer Science)

when the metallic collector is covered with an enamel layer (compare Fig. 3.33a, b)
and that fibre packing density is reduced with the increase of dielectric layer thickness
(compare Fig. 3.33b, c). This result is due to a slower charge dissipation process
occurring when a thicker enamel coating is used, that results in higher repulsive
electrostatic forces between the incoming charged fibres and in higher porosity.
The use of the above described enamel material did not induce formation of
patterned mats, on the contrary, fibres were collected in the common random
configuration. In order to obtain a pattern, the electrical properties of the enamel
3.2 Porous Scaffold Fabrication by Electrospinning 77

Fig. 3.34 SEM micrographs (two different magnifications) of P(L)LA star-patterned ES mat
deposited on enamelled collector containing montmorillonite clay particles (Reprinted from [78],
Copyright (2010), with permission from Springer Science)

were locally modified by doping it with proper additives, with the aim of affecting
the electrostatic charge diffusion and charge distribution on the collector during
the ES process. When montmorillonite clay particles were randomly distributed
into the vitreous matrix during collector fabrication,3 a ‘‘star-shaped’’ pattern was
observed on the ES polymer mat deposited (Fig. 3.34). This finding can be
associated with the high electrical mobility of the alkaline metals ions present in
the montmorillonite, which locally decrease electrical resistivity of enamel.
In correspondence to these conducting points the charge density r is low.
Therefore, they act as ‘‘potential wells’’ and aggregation centres for the incoming
nanofibres, thus leading to a ‘‘star-shaped’’ deposition.
The second type of collector enabling the obtainment of patterned non-woven
mats is composed of: (a) a metallic substrate, (b) regularly spaced metallic pins
connected to the grounded metallic substrate and (c) an insulating material laying
on the top of the metallic substrate. Figure 3.35 illustrates the target assembly.
Figure 3.36b shows the picture of a non-woven mat detached from the ‘‘pin
array’’ target reported in Fig. 3.36a. Pins were positioned on the vertexes of an
imaginary square (side length D = 5 mm). In correspondence to the positions of
the pins, the mat is thicker because fibres preferentially deposit on the conducting
pins. Fibres partially deposit on Teflon sheet, in particular they accumulate both on
the sides and on the diagonals of the square, i.e. along lines connecting the con-
ducting pins. Figure 3.36c sketches the peculiar macroscopic pattern of the
obtained mat. SEM micrographs of Fig. 3.36 show the mat micro-pattern: fibres
tend to spun across the metallic pins, uniaxially aligning along the square sides and

3
Enamel raw material was prepared by milling the frit in water for 4 h and by adding, during the
milling process, a montmorillonite clay (Mullenbach & Thewald GMBH, Germany, particle size
ca. 50 lm) as doping material. The wet blend containing the suspended clay particles was
sprayed over the steel sheet and dehydrated. Enamelled collector with a coating thickness of
0.2 mm was obtained. The collector was fired in a radiant furnace with oxidizing atmosphere at
850 C for 6.5 min.
78 3 Results and Discussion

Fig. 3.35 Schematic representation of collectors made of an array of metallic pins (d = 2 mm,)
connected to a metallic substrate, placed on a insulating Teflon sheet. a side view, b front view
and c picture of a collector where D = 10 mm

generating a cross array in the centre of the square. The collector used allows to
locally orient fibres in different directions, depending on the location of the con-
ducting pins. The control of fibre deposition is achieved, in this case, thanks to the
action of the electrical field generated between the metallic needle ejecting the
polymer solution and the pin array collector. In order to explain its effect, the study
by Liu et al. [73] is particularly useful. The authors modelled the electrical field
generated by a collector made of two metallic strips separated by an air gap. They
found that fibres uniaxially aligned perpendicularly to the strips thanks to the
electrical field component parallel to the collector surface that was responsible of
stretching the incoming fibres across the conducting strips. Authors pointed out
that, when a metallic plate is used, the electrical field does not have a component
parallel to the surface collector, thus fibres deposit randomly. Analogously, the
peculiar fibre deposition on the ‘‘pin array’’ collectors can be explained according to
the electrical field model provided by Liu et al. [73]. Moreover, the pin gap distance
D has a remarkable effect on fibre alignment degree. Indeed, by increasing pin
separation from 5 mm up to 30 mm, fibre alignment on the sides of the square was
enhanced. This result was also found by Liu et al. [73] in the case of metallic strips
separated by an air gap. However, the increase of pin distance gradually reduced the
fibre deposition on the diagonals of the square, until the cross array fibre mor-
phology in the centre of the square was not generated anymore. Moreover, at even
higher pin separation, also fibre deposition across the side of the square is reduced,
probably because the electrical field component parallel to the collector surface is
too low to stretch the incoming fibres across the conducting pins.

3.2.5 ‘‘Composite’’ Electrospun Scaffolds

Porous scaffolds composed of different types of fibres can be easily produced by

concomitantly electrospinning two different polymer solutions. To this aim, the ES
3.2 Porous Scaffold Fabrication by Electrospinning 79

Fig. 3.36 a Pin array collector (d = 2 mm, D = 5 mm), b P(L)LA ES mat produced by using
the pin array collector, c schematic representation of the macroscopic mat pattern, d, e and f SEM
micrographs of differently oriented fibres at different mat positions

instrumental apparatus was modified by using two syringes connected to two

distinct metallic needles, both positively charged, that were positioned on the
opposite sides of the grounded cylindrical collector in order to address the ejected
polymer jets on the same collector (see Fig. 3.37). The rotation of the cylinder
ensures a homogenous distribution of the two different fibres on the entire mat.
80 3 Results and Discussion

A plane collector can be employed as well. In this case, however, the two needles
are commonly vertically placed on the target that must translate in order to
homogenously collect the fibres [81].
By using the instrumental apparatus sketched in Fig. 3.37, the following
‘‘composite’’ scaffolds were produced:
• scaffold made of a single polymer type but consisting of fibres with different
morphologies. Figure 3.38a reports SEM micrographs of a P(L)LA scaffold
composed of both micrometric and sub-micrometric fibres, as an example.
• scaffold made of two different polymers. Figure 3.38b shows a mixed scaffold
consisting of P(L)LA fibres (thicker ones) and Poly(ethylene oxide) (PEO) fibres
(thinner ones).
‘‘Composite’’ ES scaffolds can be also composed by layers of different fibres
obtained by consecutively electrospinning distinct polymer solutions on the same
target [81, 82].

Fig. 3.37 Scheme of ES apparatus for the production of ‘‘composite’’ scaffolds

Fig. 3.38 a ‘‘composite’’ ES scaffold obtained by concomitantly electrospinning P(L)LA 13% w/V
n DCM/DMF (65/35) and P(L)LA 20% w/V in CLF:DMF (90/10) (DV = 14 kV, R = 0.02 ml/min,
d = 20 cm); b ‘‘composite’’ ES scaffold obtained by concomitantly electrospinning P(L)LA 13% w/
V in DCM/DMF (65/35) and PEO 6% w/V in H2O (DV = 18 kV, R = 0.02 ml/min, d = 20 cm)
3.2 Porous Scaffold Fabrication by Electrospinning 81

ES scaffolds consisting of different polymers and/or morphologies integrate,

in the same product, the specific properties of each component. Thus, it is possible
to design suitable products for the specific applications by combining fibres, that
individually taken, do not display the desired mechanical properties, affinity
towards cells and degradation rate.

3.2.6 Electrospun Scaffold Functionalization

3.2.6.1 Bulk Functionalization

One of the great advantages in using ES technique is the possibility to incorporate

additives within fibres in a one-step process. This opportunity has been extensively
exploited to load fibres with biomolecules that were intended to be released upon
contact with the biological environment. Among the several drugs employed,
antibiotics [83–85], anti-inflammatory drugs [86–88] and anti-cancer drugs [89]
have been mainly investigated.
In the present research, given the key role of GFs in addressing cell behavior, an
Endothelial Cell Growth Factor Supplement (ECGS) from Bovine Neural Tissue
(Sigma–Aldrich) was chosen as bioactive additive. In the literature other types of
GFs have been employed in combination with ES scaffolds. However, most of
these studies concern the immobilization of such biomolecules at the fibre surface
[90–92]. Leong et al. incorporated GFs that were intended to promote nerve
regeneration into ES fibres of caprolactone-ethylethylene phosphate copolymer
[93, 94]. In other studies, GF have been incorporated into fibres by using coaxial
ES [95, 96].
In the present research, ES mats of PLA50GA50 were fabricated from a 15%
w/V polymer solution in CLF/DMF/HFIP (60/30/10 by volume)4 Sub-micrometric
bead-free fibres with a diameter distribution of 790 ± 140 nm were obtained.
ECGS in powder was added to similar polymer solutions in order to yield, upon
solvent evaporation, ES PLA50GA50 mats containing two different amounts of
ECGS: 0.4 and 4.8 wt%. The addition of such amounts of ECGS to the polymer
solution does not remarkably affect fibre morphology in the ECGS-loaded scaf-
folds. ES mats supplemented with ECGS, as well as plain polymer mats not
containing ECGS, were exposed to a culture of Mesenchymal Stem Cells, derived
from human bone marrow, in order to evaluate the effect of GF on cell behavior
and to assess if it retains its bioactivity once release in the culture medium [97].
The results of these experiments will be presented in ‘‘Cell culture experiments’’ of
this chapter.

4
ES process conditions: DV = 18 kV, R = 0.02 ml/min, d = 12 cm, T = 22 ± 2 C,
RH = 45 ± 5%.
82 3 Results and Discussion

3.2.6.2 Surface Functionalization

Modification of biomaterial surface is commonly aimed at improving cell-

biomaterial interaction and at reducing the negative effects of the foreign material
in the organism. Therefore, one of the recent targets in scientific community is to
change and to regulate the surface properties of ES fibres. The scientific literature
investigating the possibility to modify ES fibre mats has been summarized in a
recent review by Yoo et al. [98]. Several approaches have been followed, among
them:
1. plasma modification: a proper selection of the plasma source enables to modify
wettability and functional groups at the biomaterial surface in order to improve
cell adhesion [99–101];
2. physical absorption of biomolecules: a variety of ECM components such as
gelatin, collagen, laminin and fibronectin have been physically immobilized at
ES fibre surface, generally after pre-treatement with plasma [102, 103];
3. surface graft polymerization: covalent immobilization of biomolecules has
been performed at fibre surface after pre-treatement with plasma or UV radi-
ation [104–107].
In the present work the surface of P(L)LA ES fibres was modified through an
approach aiming at changing fibre properties by covering their surface with highly
hydrophilic polymer brushes carrying the desired functional groups. The latter can
subsequently covalently bind suitable biomolecules according to the specific
biological response to be elicited. The hydrophilic nature of the polymer brushes
should help controlling cell adhesion, by preventing non-specific protein absorp-
tion at the scaffold surface.
Investigation of cell-biomaterial interface has elucidated that cell adhesion to a
surface is always mediated by a layer of proteins covering the biomaterial. On the
contrary, a surface that does not absorb proteins cannot support cells, due to the
absence of specific peptide sequences that allow cell attachment. Thermodynamic
principles governing the adsorption of proteins onto surfaces involve a number of
enthalpic and entropic terms that have been summarized by Ratner and Hoffman
[108]. In brief, by limiting the discussion to neutral surfaces, protein-biomaterial
interaction can be described as follows (see scheme in Fig. 3.39). In solution,
proteins assume a globular structure, with hydrophobic groups at the core and
hydrophilic residues in contact with water. When a hydrophobic biomaterial is
exposed to a biological fluid containing proteins, the latter unfold and cover the
surface in order to maximize hydrophobic interaction with the device. On the
contrary, when the biomaterial is highly hydrophilic, protein absorption is an
unfavourable process, thus cell attachment does not occur.
Despite the fact that protein absorption enables cell adhesion, non-specific
absorption is in some cases undesired because it does not allow to control and
regulate cell behaviour. In this context, the increase of wettability of ES fibres with
hydrophilic polymer brushes is expected to prevent the occurrence of non-specific
protein absorption, thus making the surface protein-repellent. At a later stage,
3.2 Porous Scaffold Fabrication by Electrospinning 83

Fig. 3.39 Schematic representation of the process of cell attachment to a material surface (not
drawn to scale). Hydrophobic surfaces absorb a layer of protein from the biological fluid and
subsequently cells adhere to the surface thanks to the recognition of specific peptide sequences
located along protein chains. Hydrophilic surfaces do not interact with the proteins in solution,
thus cell attachment cannot occur

polymer brushes can be functionalized with biomolecules (e.g. specific peptide

sequences) in order to specifically control cell attachment.
The present research preliminarily investigated the possibility of modifying ES
fibres of P(L)LA. The work is based on a previous study carried out in collabo-
ration with the University of Manchester, that investigated the functionalization of
TCPS flat substrates with copolymer brushes of Glycerol Monomethacrylate
(GMMA) and 2-(dimethylamino)ethyl methacrylate by Surface Initiated Atom
Transfer Radical Polymerization (SI-ATRP) [109]. An analogous functionalization
procedure was applied in this work to a 3D P(L)LA porous scaffold. The procedure
was composed of three steps:
1. electrospinning of a P(L)LA solution containing star-branched oligo(D,L)lactic
acid possessing carboxilate terminal groups (PLA-T6), with the aim of fabri-
cating fibres with negatively charged surface upon water exposure;
2. physical absorption of a positively charged macroinitiator (MI, Fig. 3.40a) on
fibre surface, with the aim of generating sites for initiating Atom Transfer
Radical Polymerization (ATRP);
3. synthesis of polymer brushes by SI-ATRP of GMMA (Fig. 3.40b), with the aim
of producing protein-repellent fibre surfaces.
ATRP belongs to the group of the controlled radical polymerizations (CRP). The
main advantage of this kind of synthetic approaches is the possibility to polymerize
a huge number of monomers obtaining polymers with wide ranges of molecular
weights and low polydispersities. In CRP initiation reaction is fast, providing a
constant concentration of growing polymer chains. In addition, termination reac-
tions, responsible of the uncontrolled common radical polymerizations, are mini-
mized. Termination reactions are almost completely suppressed by maintaining,
during the polymerization, a very low concentration of reactive radicals.
84 3 Results and Discussion

Fig. 3.40 Chemical

structures of a ATRP MI and
b GMMA

This is obtained by using a radical species that is reversibly deactivated to a dor-

mant species. It is worth noting that the equilibrium between the dormant and the
active species is effective in controlling the polymerization if: (1) it is shifted
towards the dormant species and (2) the rate of dormant-active species exchange is
faster than the rate of propagation, so that all polymer chains have the same
probability of adding monomer [110, 111].
In ATRP the dormant species is an alkyl halide (RX, R = Br, Cl, I) and its
deactivation is promoted by the presence of a transition metal complex which
undergoes an equilibrium between two oxidation states (Mzt –Ln ¢ X–Mz+1 t –Ln).
ATRP is widely employed as a versatile technique to prepare bioactive surfaces,
including anti-fouling, antibacterial, stimuli responsive and patterned surfaces,
through brushes grafting [112]. Fu et al. applied SI-ATRP for generating polymer
brushes on fibres by electrospinning an ad hoc synthesized Br-terminated poly-
styrene [113]. Subsequently, ATRP was initiated by Br terminals present at fibre
surface.
In the present research, the MI reported if Fig. 3.40a was absorbed on P(L)LA ES
fibres through electrostatic interaction, with the aim to modify the fibre surface of
commercial ES polymers. The MI should be easily and steadily absorbed on a
negatively charged surface thanks to the cooperative effect of many positively
charged repeating units. Moreover, the large amount of Br initiator groups carried by
the MI is expected to generate densely packed polymer brushes. Negative charges can
be generated on fibre surface by electrospinning a star-branched oligomer, possessing
carboxylate terminals (PLA-T6), together with P(L)LA. The use of PLA-T6 oligo-
mers should enable a good blending between the P(L)LA matrix and the additive.
Moreover, it has been reported that, during fibre formation, the charge applied to the
jet promotes the migration of the polarizable species contained in the solution (PLA-
T6 in this case) towards the surface of the fibre [114, 115].
A solution of P(L)LA 13% w/V (DCM/DMF:65/35, by volume) containing 10%
w/w of PLA-T6 was electrospun5 and the obtained fibres had a average diameter of

5
ES conditions: DV = 16 kV, R = 0.01 ml/min, d = 15 cm, T = 24 ± 2 C, RH =33 ± 3%.
3.2 Porous Scaffold Fabrication by Electrospinning 85

435 ± 125 nm. It is worth noting that by electrospinning the same P(L)LA
solution not containing PLA-T6 additive, thicker fibres were produced (diameter
distribution 610 ± 180 nm, see Sect. 3.2.2). It is well established that the addition
of salts—in our case PLA-T6 oligomer carrying six carboxilate groups—to a
polymer solution enables the obtainment of thinner fibres thanks to the increase of
solution conductivity [43, 45, 116].
Scaffold specimens were immersed in a MI solution (0.1% w/V in water)
overnight to allow electrostatic absorption to occur. The absorption conditions
were selected according to previous results concerning absorption of the same MI
on TCPS [109].
SI-ATRP of polyGMMA brushes was carried out on MI-coated scaffolds as
described in ‘‘Materials and Methods’’. GMMA unit possesses two hydroxyl groups
that confer hydrophilic character to the polymer brushes. Previous studies reported
that GMMA is a non toxic, protein-repellent, and non cell-adhesive building block
[109, 117, 118]. Moreover, its hydroxyl groups can act as reactive sites for
subsequent desired functionalization reactions. Figure 3.41 compares fibre mor-
phologies of P(L)LA scaffolds loaded with PLA-T6 at different stages of the func-
tionalization procedure. Figure 3.41a reports SEM micrograph of the starting
sample, Fig. 3.41b shows the scaffold after immersion in MI solution and Fig. 3.41c
shows fibres after SI-ATRP synthesis of polyGMMA. Fibre surface was also
explored at higher magnifications than that reported in Fig. 3.41 and no evident
differences in fibre morphology were detected by SEM after the different treatments.
In order to verify the occurrence of MI absorption and of polyGMMA brushes
growth at the fibre surface, electrokinetic analyses were performed. In general,
when a charged solid surface comes into contact with a water solution, the free
charges in solution distribute at the interface and they generate an electrochemical
double layer. The latter is commonly divided into an immobile layer close to the
solid surface and a mobile one. The potential at the interface of these two layers is
called f-potential and its value is related to the charge density on the solid surface.
f-potentials of the starting P(L)LA scaffolds, of samples after immersion in MI
solution and of scaffolds after SI-ATRP were measured at pH values in the range
5–9 (see Fig. 3.42). Surface charge density of ES samples is highly affected by
both MI and SI-ATRP treatments. At pH = 6 the f-potential of the starting sample
was around -60 mV as a consequence of the presence of negative charges on the

Fig. 3.41 SEM micrographs of P(L)LA fibres loaded with PLA-T6: a starting sample, b after MI
treatment and c after SI-ATRP of GMMA
86 3 Results and Discussion

surface provided by the carboxylate groups of PLA-T6. After immersion in MI

solution, the f-potential at pH = 6 increased to -20 mV. This finding can
be explained by assuming that MI partially neutralizes the negative charges at the
fibre surface, demonstrating that MI absorption occurs. After SI-ATRP the
f-potential was 0 mV over the entire pH range investigated. Such a f-potential
trend is explained assuming that a layer of neutral polyGMMA brushes covers
fibre surface. Since hydroxyl groups in polyGMMA are very weak acids, the
surface is neutral (f-potential = 0) over the entire range of pH investigated
(pH = 5–9).
The successful modification of ES fibre surface was definitely confirmed by
qualitative wettability tests. As shown in Fig. 3.43a, the starting sample is not
wetted by water given both the intrinsic hydrophobic nature of P(L)LA and the
presence of air entrapped within the pores of the scaffold. On the contrary, after the
SI-ATRP procedure, the sample is instantaneously wetted by water (Fig. 3.43b).

Fig. 3.42 Electrokinetic data

of ES samples differently
treated

Fig. 3.43 Representative pictures of: a highly hydrophobic ES starting sample and b wet ES
sample after polyGMMA SI-ATRP
3.2 Porous Scaffold Fabrication by Electrospinning 87

It is worth noting that mat 3D porous structure was not affected by the ATRP
polymerization (see Fig. 3.41c), hence the change of mat wettability is ascribed to
the presence of hydrophilic polyGMMA brushes covering fibre surface.
The preliminary results of fibre surface modification described above open a
wide range of possibilities that, for a lack of time, haven’t been attempted in the
course of the present Ph.D. Indeed, the surface passivation towards protein
absorption, and thus towards cell attachment, is the basic requirement to achieve a
good control of cell behaviour. Future work will aim at exploiting the hydroxyl
groups of polyGMMA brushes for immobilizing biomolecules, such as peptides or
GFs, for eliciting the desired cell behaviour.

3.3 Hydrolytic Degradation Experiments

Scaffolds that are intended to maintain their role and functionalities for a limited
period of time must inevitably undergo a process that leads to their gradual
disappearance. Cell supports generally disappear as a consequence of a degra-
dation process which cleaves the macromolecular chains. Sometimes these
reactions are enzyme-catalyzed in vivo but usually polymer degradation process
involves a simple hydrolytic cleavage of the chain bonds with a consequent
reduction of chain length and of dissolution of water-soluble short molecules.
The relative rates of water uptake and bond cleavage determine the degradation
mode: when water uptake is faster than hydrolysis the process involves the entire
sample (‘‘bulk’’ degradation), when, on the contrary, water diffusion into the
sample is slower than hydrolysis, surface erosion occurs. The first degradation
mode is typical of polyesters while the latter is common to polyanhydrides [119].
Polyesters are the only class of materials employed in the course of the present
research, therefore the following discussion strictly concerns the mechanism of
‘‘bulk’’ degradation.
To date, hydrolytic degradation studies carried out in vitro at physiological
conditions have elucidated that polyester scission rate depends on a number of
factors, e.g. chemical structure, morphology (amorphous to crystalline phase ratio)
and specimen dimensions [120]. Chain hydrophilicity is the most important
parameter affecting degradation. For poly(x-hydroxyacids), hydrophlicity depends
on the ratio between the number of ester groups and the number of carbon atoms
along the chain. The rate of water uptake depends also on phase morphology:
a densely packed crystalline phase is less permeable to water than the amorphous
phase. Degradation can occur over several years for highly hydrophobic semi-
crystalline polymers, such as PCL, or within few weeks for more hydrophilic
amorphous materials, such as PLA50GA50. Another key parameter that affects the
degradation process is specimen dimension which determines the occurrence of
autocatalysis. Acidic autocatalysis is due to the presence of carboxylic end groups
on chain fragments generated as a consequence of ester cleavage [120]. They
decrease the pH in the surroundings with a consequent increase of hydrolysis rate.
88 3 Results and Discussion

Fig. 3.44 DSC first scans of

as-spun P(L)LA (solid line)
and ES P(L)LA after EtOH
treatment (dotted line). Insert:
ES P(L)LA soaked in EtOH

Autocatalysis mostly affects the degradation rate of large specimens, owing to the
limited diffusion of the acidic polymeric fragments that accumulate at the speci-
men core. In these cases, the degradation rate is faster in the bulk than at the
sample surface.
Given the great importance of resorbability time-scale in the body, degradation
mechanism of polyesters has been intensively studied in vitro, although, in the
organism, other factors interfering with the degradation process can come into
play. However, the in vitro experiments can help hypothesizing the kind of deg-
radation mechanism and rate of a certain device in vivo.
In the present research, in vitro hydrolytic degradation experiments were per-
formed on semicrystalline P(L)LA ES scaffolds. P(L)LA is universally recognized
as a bioresorbable material and it is largely employed in biomedical applications.
Its final degradation product is lactic acid, which is metabolized by the organism
through the Krebs cycle.
Non-woven mats obtained by electrospinning a solution of P(L)LA 13% w/V in
DCM/DMF (65/35 by volume)6 were completely amorphous, as previously dis-
cussed in Sect. 3.2.2. In order to induce crystallization, as-spun fibres were
immersed in EtOH. The alcohol plasticizes P(L)LA, whose Tg decreases from
60 C down to 22 C (by DSC analysis of as-spun P(L)LA soaked in EtOH,
Fig. 3.44 insert). As a consequence, polymer crystallization window enlarges
towards lower temperatures. In order to induce polymer crystallization, ES P(L)LA
samples were therefore kept in EtOH overnight at RT. EtOH exposure did not
induce any evident morphological changes to the ES fibres, which maintained their
orientation and diameter distribution. Figure 3.44 shows DSC curve of ES P(L)LA
after EtOH exposure overnight at RT together with DSC curve of the as-spun
amorphous P(L)LA for comparison. In the latter the cold crystallization exotherm

6
ES conditions: DV = 15 kV, R = 0.02 ml/min, d = 15 cm, T = 22 ± 2 C, RH = 41 ± 3%.
3.3 Hydrolytic Degradation Experiments 89

peak is followed by a melting endotherm of the same entity. On the contrary, after
EtOH treatment, DSC curve of ES P(L)LA displays a broad cold crystallization
peak followed by a melting endotherm of higher entity, revealing the occurrence of
crystallization upon EtOH exposure.
Hydrolytic degradation experiments on semicrystalline P(L)LA ES mats were
run at 37 C in buffer solution (pH = 7.4) up to a maximum of 396 days. After
selected exposure times, the samples were recovered from solution and, after
washing and drying, they were subjected to gravimetric, GPC, WAXS, DSC and
SEM analyses. It is pointed out that buffer solution was periodically changed in
order to maintain the samples at neutral pH, thus reproducing the physiological
buffered environment. Samples are labelled X-P(L)LA, where X represents the
days of permanence in buffer solution.
The GPC elugrams of ES samples recovered after selected hydrolytic degra-
dation times are shown in Fig. 3.45, together with the GPC elugram of the unde-
graded sample (0-P(L)LA) as a reference. Over the whole time scale of the
experiment, the P(L)LA macromolecules gradually eluted at higher retention vol-
umes, thus indicating a progressive shift towards lower molecular weights. This
result shows that the samples recovered from buffer after increasing exposure times
contained a growing fraction of low molecular weight chains and a smaller number
of long macromolecules. It is worth nothing that the elution curve, not only shifted
towards lower molecular weights, but it also changed its shape. The initial elugram
(sample 0-PLLA) had a strong peak corresponding to a molecular weight value of
about 126,000 g/mol (green arrow in Fig. 3.45). The elugram of the starting sample
also displayed a second weak peak in correspondence to a molecular weight of
6300 g/mol (red arrow in Fig. 3.45). In the course of degradation, the peak at higher
molecular weights decreased and shifted towards higher elution volumes, while
the peak at lower molecular weights gradually increased in the course of the

Fig. 3.45 GPC elugrams of

P(L)LA mats retrieved from
buffer after selected exposure
time
90 3 Results and Discussion

experiment, without changing its position. After about 290 days of buffer exposure,
a strong third peak, corresponding to a molecular weight of 3100 g/mol, appeared
(blue arrow in Fig. 3.45, sample 293-P(L)LA, 326-P(L)LA and 396-P(L)LA).
By integrating the GPC elugrams shown in Fig. 3.45, the molecular weight
values reported in Table 3.5 were obtained. The Table lists both weight (Mw) and
number average molecular weight (Mn) together with the percent of sample mass
remaining after buffer exposure, m(%).
Figure 3.46 compares the changes of sample weight as a function of time with
the corresponding mass changes at the molecular level (Mw changes), both
quantities being expressed as percentage remaining after a given time in buffer
solution. It is pointed out that, since the shape of the molecular weight distribution
changed in the course of the degradation (see Fig. 3.45), Mw only provides an
overall information of the change of polymer molecular weight as a consequence
of hydrolysis. The molar mass significantly decreased from the very beginning of
the degradation experiment, while the ES mats showed a very slow weight loss.

Table 3.5 Gravimetric and GPC results on hydrolytically degraded electrospun P(L)LA samples
Sample Exposure time (days) Mw (g/mol)a Mn (g/mol)a m (%)b
0-P(L)LA 0 172320 52340 100
65-P(L)LA 65 98310 14890 93.7
159-P(L)LA 159 81390 17680 91.9
231-P(L)LA 231 49860 8560 82.9
293-P(L)LA 293 32794 4975 80.1
326-P(L)LA 326 20600 3380 n.d.c
396-P(L)LA 396 19310 2740 n.d.c
a
from GPC
b
percentage weight remaining after buffer exposure, calculated according to Eq. 2.1
c
n.d. = not detectable due to loss of sample integrity

Fig. 3.46 Weight percentage

remaining (m%) (filled circle)
and molar mass percentage
remaining (Mw%) (filled
triangle) as a function of
buffer exposure time
3.3 Hydrolytic Degradation Experiments 91

The observed weight loss is attributed to dissolution into the surrounding medium
of low molecular weight chain fragments produced by hydrolytic degradation.
It is pointed out that the weight loss was difficult to detect with the advancing
of degradation. In the final part of the experiment (samples 326-P(L)LA and
396-P(L)LA) weight loss was not measurable anymore, due to sample fragility and
loss of integrity. It is interesting to note that around day 290 the rate of molecular
weight decrease abruptly slowed down. This result can reflect the absence, in
samples recovered after more than 290 days in buffer, of the short chain fragments
produced by hydrolysis, which were responsible of the fast Mw decrease up to day
290. For exposure times longer than 290 days, such fragments were likely released
from the sample via water solubilization.
WAXS analysis were performed on P(L)LA ES samples recovered after dif-
ferent buffer exposure times, in order to evaluate changes in the crystal phase
during degradation. Figure 3.47a reports diffractograms of selected samples. The
two crystalline reflection peaks at 2h = 16.5 and 19 are typical of the a-form of
P(L)LA having a pseudo-orthorhombic unit cell [121] and their intensity increased
with the progress of degradation. Figure 3.47b plots the crystallinity degree—
calculated as the ratio of the crystalline peak areas to the total area under the
scattering curve—as a function of degradation time and shows that the degree of
crystal phase sharply increased after around 230 days. It is pointed out that, at a
similar time scale of degradation, the decrease of average molecular weight,
previously reported in Fig. 3.46, slowed down as a consequence of low molecular
weight chains dissolution. Therefore, it is reasonable to assume that degradation
mostly occurred in the amorphous phase and that the amount of crystal phase
mainly increased as a consequence of macromolecules released from the amor-
phous phase. However, crystallization of part of amorphous molecules during
buffer exposure can not be excluded.
DSC curves of selected P(L)LA samples after different exposure times are
reported in Fig. 3.48. The single melting peak at Tm = 160 C (sample 0-P(L)LA)

Fig. 3.47 WAXS data: a diffractograms of selected P(L)LA samples retrieved from buffer after
selected exposure times and b change of crystallinity degree in the course of degradation
experiment
92 3 Results and Discussion

Fig. 3.48 DSC first scans of

selected P(L)LA samples
after different times of
buffer exposure (heating rate
20 C/min)

shifted and broadened toward lowers temperature with the advancing of degra-
dation (samples 65-P(L)LA, 231-P(L)LA and 293-P(L)LA). Interestingly, last
samples retrieved from the buffer (i.e. samples 326-P(L)LA and 396-P(L)LA)
displayed a melting endotherm characterized by an additional peak at low tem-
peratures (Tm = 145 C).
SEM observations of selected ES mats retrieved during the degradation exper-
iment show a gradual change of fibre morphology (Fig. 3.49). After 230 days of
degradation, fibres appeared broken in some points (Fig. 3.49b) and the last two
samples retrieved from the buffer (326-P(L)LA and 396-P(L)LA) showed many
broken fibres. Moreover, the initial smooth fibre surface (see Fig. 3.49a) became
rough as a consequence of the degradation (see inserts in Fig. 3.49c, d). As previ-
ously discussed, the measured of weight loss was impossible for samples
326-P(L)LA and 396-P(L)LA due to their fragility and fragmentation (see m%
values in Table 3.6). Therefore, it cannot be excluded that fibres shown in Fig. 3.49c
and d might have been broken during preparation of the sample for SEM analysis.
Gravimetric, GPC, WAXS, DSC and SEM analyses, taken together, provide
information about the hydrolysis of semicrystalline P(L)LA ES fibres. As previ-
ously mentioned, the slowdown of the decrease of Mw occurring around 290 days
of degradation (by GPC, see Fig. 3.46), coupled with the increase of crystallinity
degree at a similar time scale (by WAXS, see Fig. 3.47), revealed that hydrolysis
mostly occurred in the amorphous phase. Particularly significant is the change of
molecular weight distribution in the course of degradation. The shape of GPC
elugrams reported in Fig. 3.45 can be explained referring to Vert’s thorough studies
concerning hydrolytic degradation of polylactide thick films. Vert et al. ascribed the
formation of multimodal molecular weight distributions to the presence of mac-
romolecular chains degrading with a different rate in the course of the experiments,
i.e. (1) macromolecules in amorphous phase that degrade faster than macromole-
cules in the crystal phases and/or (2) macromolecules in specimen core which
degrade faster than those at the surface, as a consequence of the autocatalytic effect
3.3 Hydrolytic Degradation Experiments 93

Fig. 3.49 SEM micrographs of: a 0-P(L)LA, b 231-P(L)LA, c 326-P(L)LA and d 396-P(L)LA

[122–124]. When the authors studied thick specimens of completely amorphous

polymers, the molecular weight distributions displayed two distinct peaks, only as a
consequence of the autocatalysis. In the case of semicrystalline thick films, the
autocatalytic effect combined with the different degradation rates of amorphous and
crystalline phase led to multi-peak molecular weight distributions. In the case of ES
materials, the autocatalysis is unlikely given the extremely small fibre section. This
point was confirmed by a previous study investigating the hydrolytic degradation of
completely amorphous PLA50GA50 ES fibres [97].
The progressive change of semicrystalline ES P(L)LA molecular weight
distribution (see Fig. 3.45) in the course of degradation experiment might be
interpreted as follows. At the initial stages degradation interests macromolecules
in the amorphous phase. The peak corresponding to the high molecular weight
fraction (MW = 126,000 g/mol, green arrow in Fig. 3.45) shifted towards lower
molecular weight values and it decreased, while the low molecular weight fraction
peak increased (MW = 6300 g/mol, red arrow in Fig. 3.45). The latter did not
change its position probably because the molecules produced by hydrolysis having
molecular weights lower than 6300 g/mol dissolved in the buffered medium, thus
they were not detectable by GPC analysis of the solid recovered sample. With the
proceeding of degradation, after 290 days of buffer exposure, the appearance of a
further peak at higher elution times (MW = 3100 g/mol, blue arrow in Fig. 3.46)
reveals the presence of very low molecular weight fragments which cannot
dissolve in water. The appearance of this third peak in the GPC elugram can be
94 3 Results and Discussion

explain as follows. Water does not penetrate the densely packed crystal phase, but
it has access to crystal surface, where it can cleave ester bonds located on folded
amorphous chain portions. However, if other portions of the same macromolecule
are blocked in the crystal phase, the chain cannot dissolve in the aqueous
environment. Therefore, these low molecular weight fragments blocked in the
crystal phase can be detected by GPC analysis of the recovered solid samples.
This hypothesis might also explain DSC results previously reported in Fig. 3.35.
Indeed, with the proceeding of degradation, DSC curves showed the appearance, in
the melting endotherm, of an additional peak at low temperatures (Tm = 145 C)
which may be associated with the melting of short chain fragments.
This study provides data on the time-scale of in vitro hydrolytic degradation of
semicrystalline ES P(L)LA mats. Results demonstrated that, despite the polymer
molecular weight started to decrease from the very beginning of the experiment,
weight loss was very slow within 10 months (290 days) of buffer exposure. After
this period of time, the sample became fragile and loss its integrity, thus loosing its
structural functionality. This can lead to the release of small pieces of fibres in the
body which can be phagocytized by foreign body-giant cells [125, 126]. Such kind
of information is crucial in the design of scaffolds for TE that should maintain their
mechanical performance for a given period of time after which they should be
resorbed.

3.4 Cell Culture Experiments

In the course of the present Ph.D., ES scaffolds, designed, fabricated and characterized
as described in the previous chapters, were employed in cell culture experiments
performed in collaboration with biochemical laboratories. The contribution of
material science was precious in the context of these interdisciplinary collaborations.
Indeed, a deep knowledge of polymer chemical-physical properties is a necessary
requirement, not only to design and fabricate ES products, but also to improve stan-
dard cell culture operating procedures in order to perform biological experiments by
using unconventional cell substrates, i.e. ES scaffolds.
Cell culture experiments illustrated in this chapter were carried out in collab-
oration with the Biochemistry Department ‘‘G. Moruzzi’’ (University of Bologna),
the Clinical Department of Radiological and Histocytopathological Sciences
(University of Bologna) and the Foundation for Development of Cardiac Surgery
(Zabrze, Poland).

3.4.1 Scaffold Preparation for Cell Culture Experiments

Prior to any cell culture experiment, scaffold sterilization is a necessary step for
preventing bacterial and fungi contamination and it is even more important for
hindering the transmission of infectious pathogens to patients when implantable
3.4 Cell Culture Experiments 95

devices are used. Common biomaterial sterilization techniques are: (1) treatment
with ethylene oxide (EtO) vapour, (2) thermal treatment, (3) irradiation with UV
light or (4) with c-rays.
EtO treatment is highly recommended for biomaterial sterilization. However,
this substance is highly toxic and inflammable, thus sterilization is often performed
by specialized companies at high costs. Moreover, since EtO sterilization is often
performed around 50–60 C, its use is not recommended for polymers with Tg
values in this temperature range (e.g. PLA50GA50).
Thermal treatment is usually carried out by gradually increasing water vapour
temperature up to approximately 120 C. While these conditions grant for the
death of many bacteria, they can be detrimental for polymers that melt below
120 C or that are susceptible of hydrolytic degradation.
Other sterilization procedures involve irradiation. Among them, the most
accessible method consists in using ultraviolet (UV) light which sterilizes without
inducing secondary reactions. However, the low penetration ability of UV radia-
tion makes the efficacy of this technique limited, especially for thick fibre mats.
Compared to UV light, c-rays offer a more complete sterilization since they are
able to penetrate into the irradiated materials. Possible concerns arise from
alterations of the irradiated polymer which can undergo chain cleavage, cross-
linking reactions or oxidation.
The most commonly used method for sterilizing ES scaffolds is Ethanol (EtOH)
wetting and its effectiveness has been largely demonstrated. It is pointed out,
however, that EtOH is not considered as a sterilizer but it is classified as a dis-
infectant, being effective against bacteria, fungi and many viruses but not against
bacterial spores. ES meshes are simply immersed in EtOH for 15–30 min and
extensively washed before use, typically with phosphate buffered saline solution or
cell culture medium added with antibiotics. A great advantage in using EtOH is
that it allows to wet the intrinsically hydrophobic mats because it spontaneously
enters the pores of the structure. However, EtOH sterilization also has a drawback
because some ES mats are not dimensionally stable when immersed in EtOH. Such
scaffolds may undergo a macroscopic shrinkage that is accompanied by micro-
scopic changes of fibre morphology: fibres become curly, fibre diameter increases
and pore size decreases. This behaviour has been reported for some polymers
such as PLA50GA50 or P(D,L)LA, whereas other materials such as semicrystalline
PCL do not undergo extensive shrinkage in the same experimental conditions
[83, 127, 128].
In ES scaffolds, the macroscopic shrinkage and the change of fibre morphology
has been attributed to changes of molecular conformation due to chain relaxation
occurring when macromolecules in the amorphous state acquire mobility [128].
Indeed, when the fibre is generated during the ES process, polymer chains are
stretched in the fibre axis direction, while the solvent quickly evaporates. If the
stretched molecular chains do not have enough time to undergo relaxation before
complete solvent evaporation, they will solidify in an elongated conformation.
Afterwards, if for any reason macromolecules acquire mobility at a temperature
close to or higher than their Tg, they will change their conformation from the
96 3 Results and Discussion

stretched oriented one towards the thermodynamically stable random coil one.
EtOH acts as a plasticizer for many of the polyesters commonly employed in TE.
Therefore, if the presence of EtOH leads to a decrease of polymer Tg below RT,
the shrinkage can occur during the sterilization procedure at RT.
The effect of EtOH on fibre morphology is clarified by the following example.
A square ES mat (30 9 30 mm) of P(LA-TMC) was placed in EtOH for 1 h at
room temperature. The Tg of ES P(LA-TMC) is around 34 C and it decreases
down to 0 C when the mat is immersed in EtOH (by DSC analysis of as-spun
P(LA-TMC) fibres soaked in EtOH, DSC curve not shown). Figure 3.50 shows the
effect of EtOH on random P(LA-TMC) fibres together with the percentage of
shrinkage calculated as:
‘  ‘0
s¼  100 ð3:1Þ
‘0
Where ‘ is the side length of the mat after EtOH treatment and ‘0 is the initial
side length (30 mm). After 1 h in EtOH, fibre morphology changed: fibre diameter
increased, fibres became more packed and pore dimension decreased (compare
Fig. 3.50a with b). As already pointed out, this finding can be attributed to chain
relaxation occurring during EtOH treatment. Moreover, when patterned mats or
mats made of aligned fibres are placed in EtOH they loose completely their ori-
ginal fibre orientation. These changes of scaffold dimension and of fibre mor-
phology introduce obvious limitations in the use of those ES materials that
undergo strong shrinkage during their sterilization and some authors have even
excluded the use of such polymers for TE applications [83, 127, 128].
This drawback has been circumvented in the course of this research by a
procedure that allowed the macromolecules to relax, while preventing shrinkage.
With this aim in mind, the ES scaffold was attached to a rigid plastic frame and
placed in EtOH. Being the mat bound to the frame, its gross dimensions did not
change and fibres tended to maintain their morphology (compare Fig. 3.50a with c).
After this ‘‘constrained’’ pre-treatment in EtOH, the scaffold was removed from the
frame and it was immersed again in EtOH to ascertain whether any dimensional
changes occurred in this second wetting step. Figure 3.50d shows that, although
some very limited shrinkage still occurred, fibre morphology was maintained even
if the scaffold was not constrained anymore.
As already pointed out, when P(LA-TMC) mats are immersed in EtOH, mac-
romolecules undergo transition from the frozen, glassy state to the mobile state.
Therefore, results obtained using the above described ‘‘constrained’’ treatment
may be interpreted as follows. During the ‘‘free’’ EtOH treatment (i.e. when the
scaffold was not ‘‘constrained’’ by the rigid frame) macromolecules relaxed from
the stretched conformation and underwent spontaneous coiling. A change of fibre
morphology and mat dimensions was therefore observed (Fig. 3.50b). These
changes did not occur when the mat was immersed in EtOH after fixing it to a rigid
frame (‘‘constrained’’ treatment) (Fig. 3.50c). It should be pointed out that also in
this case chain relaxation phenomenon occurred (i.e. macromolecules partially
changed their conformation from the aligned to the entropically favoured coiled
3.4 Cell Culture Experiments 97

Fig. 3.50 Effect of EtOH treatments on P(LA-TMC) fibre morphology. s is the percentage of
shrinkage calculated according to Eq. 3.1

one) but chains also tended to flow one respect to the other, since the constrained
fibres had fixed length and they could not macroscopically follow the change
of molecular conformation. Afterwards, when the scaffold was immersed again in
EtOH, without any constraint, only some residual shrinkage was observed
(Fig. 3.50d), that is attributed to a minor fraction of chains still in the stretched
oriented conformation. It is reasonable to assume that a longer ‘‘constrained’’
treatment in EtOH would have allowed to completely eliminate any residual
shrinkage. In conclusion, the ‘‘constrained’’ treatment performed before scaffold
sterilization in EtOH can be an effective way to limit or totally eliminate scaffold
shrinkage and fibre morphology changes and may broaden the range of ES
polymers that can be employed for TE applications, using EtOH sterilization.
98 3 Results and Discussion

In this work, with the aim to prevent scaffold shrinkage and to allow an easy
handling of ES scaffolds during cell culture experiments, ES mats were therefore
attached to plastic rings (Tecaflon PVDF) by using non-toxic silicone (see
Materials and Methods). A similar commercial solution is alternatively offered by
Scaffdex, a company that sells ‘‘crown rings’’ to immobilise thin membranes.
These inserts are typically custom-made in order to fit in common culture well-
plates with given dimensions (see Materials and Methods). Both solutions allow to
easily handle ES mats which are typically thin and consist of extremely light
fibrous networks that easily twist, wind up, or fold. Scaffold fixation on plastic
rings also avoids cell dispersion/outflow during cell culture experiments by
obtaining a cell-leakage-proof well that can be employed to carry out quantitative
cell culture experiments.

3.4.2 Characterization of Cells Cultured on Electrospun

Scaffolds

According to TE approach, initially cell culture is carried out in vitro and the
implantation of the cell-scaffold construct comes after a variety of cell assays
aiming at characterizing and identifying the behaviour of cells in contact with the
synthetic scaffold. To this aim, scientific community has successfully applied some
of 2D cell culture characterization tests to 3D cell cultures, without any substantial
modification of the protocols. However, it is sometimes impossible to characterize
cells without adapting the specific assay to the specific scaffold used.
To date, the behaviour of cells cultured on ES scaffolds is investigated mainly
through the following techniques:
• MTT, MTS or Alamar Blue colorimetric assays for the evaluation of cell via-
bility and proliferation [39, 129–131]. They exploit the change of absorption
spectra of a chromophore as a consequence of its reduction by cell reductase
enzymes. Since reduction takes place only when enzymes are active, these tests
measure the metabolic activity of viable cells which provides an indirect
information on cell number.
• Real Time Polymerase Chain Reduction (PCR) for determining the abundance
of different RNA molecules within the cells in order to assess regulation of gene
expression [130, 132–134].
• Western Blotting for semiquantative detection of specific protein expression
[132, 134, 135]. After cell detachment from ES scaffold, proteins are extracted
from cells and they are separated by using gel elecrophoresis.
• SEM observations which represent the gold standard for the evaluation of cell
morphology.
Histochemical and immunohistochemical techniques, which are normally
employed for the investigation of cells in native tissues, are not largely used for
the characterization of in vitro cell cultures on 3D scaffolds, despite they provide
3.4 Cell Culture Experiments 99

unique information to identify cell behaviour when analysing native tissues.

These techniques consist in observing thin slides of tissue under light microscope.
Staining of nuclei and cytoplasm (histochemistry) or of proteins, carbohydrates
and lipids (immunohistochemistry) with specific antibodies enables to visually
investigate cell behaviour and metabolism. When these techniques are applied to
the study of cells grown in vitro on 3D scaffolds, the preparation of the sample
and the overwhelming difficulties in obtaining good sections of the porous
structure have limited their use. Indeed, it has been reported that aliphatic
polyesters (the main constituent materials of bioresorbable scaffolds) are very
sensitive to common processing for the preparation of the sample for histo-
chemical and immunohistochemical analysis, which is believed to lead to scaffold
artifactual modifications [136–138]. As a matter of fact, common protocols
involve the use of temperatures higher than RT and organic substances which
might be solvents for the scaffolds.
The cryosection procedure is a less destructive protocol largely applied in the
literature for obtaining cross-cryosections of ES samples without using organic
solvents. Histochemical staining of these cross-sections gives the possibility to
study cell infiltration into the porous structure which is a key issue for the inte-
gration of the implanted material into the recipient tissue [132, 139, 140]. How-
ever, this procedure does not provide optimal sections for histochemical and
immunohistochemical analysis since it often leads to scaffold fragmentation and
loss of cell details.
In order to circumvent the above discussed drawbacks, information similar to
that provided by immunoistochemistry can be obtained by immunofluorescence
performed directly on nanofibrous scaffolds [131, 133, 134, 141]. In this case the
antibody is tagged with a fluorophore which is visualized under the UV-light
microscope. Immunofluorescence enables evaluation of specific antigen expres-
sion, distribution of focal adhesion contacts and of cell differentiation markers.
However, major drawbacks are low spatial resolution and polymer autofluores-
cence, hence the highly expensive confocal microscopy is often needed to analyze
these 3D samples [39, 129, 130].
The limitations of the above described methods suggest that evaluation of cell
nanofibre interactions is still technically demanding. In the course of the present
Ph.D., knowledge of material and scaffold properties were fundamental in order to
correctly work with ES samples seeded with cells. Besides most of the common
analysis already employed in the literature for characterizing cells seeded on ES
scaffolds (e.g. SEM, Alamar Blue assay, Western blotting, immunofluorescence,
etc.), a new sample preparation procedure, commonly used for native tissues, was
adapted in order to perform histological and immunohistochemistry analysis on ES
samples. The protocol, applied to P(L)LA ES fibres, was based on automatized and
standardized Formalin Fixed Paraffin Embedding (FFPE) procedure. A processing
temperature around 56 C and the use of suitable organic substances that does not
dissolve P(L)LA enabled to successfully embed the scaffold in a paraffin block.
Both cross-sections and ‘‘en face’’ slices were obtained and they were subsequently
stained for conventional histological and immunohistochemical analysis [142]. The
100 3 Results and Discussion

applied protocol allowed the preparation of intact sections of P(L)LA ES fibres

without damaging the structural integrity of the scaffold. Indeed, P(L)LA has a glass
transition temperature around 61 C, thus higher than the temperature used during
the treatment with melt paraffin (T = 56 C). It should be taken into account that
polymeric biomaterials possessing transition temperatures lower than embedding
process temperature might not be successfully processed using the same protocol.
The preparation of cross and en face-sections of ES samples seeded with cells
opens the possibility to easily obtain qualitative and semi-quantitative informa-
tion—e.g. cell morphology, ECM production, cell adhesion, death and immuno-
phenotype expression—essential whenever a bioresorbable scaffold is designed for
in vivo applications. Moreover, FFPE cross-sections offers the possibility to
analyze cell infiltration providing clearer cellular details than the more common
employed cross-cryosections.
In this work, histochemical and immunohistochemical analysis, together with
the other cell characterization techniques (e.g. SEM, Alamar Blue assays, immu-
nofluorescence, etc.), were applied in the course of the cell culture experiments in
order to evaluate cell behaviour and cell-scaffold interactions.

3.4.3 Biocompatibility Evaluation of Electrospun Scaffolds:

The Example of PPDL

As previously discussed in Chap. 1, the primary requirement of any biomedical

device is to be biocompatible. Therefore, whenever a new device is used in
contact with a biological environment, biocompatibility is imperatively the first
characteristic to be verified. Unfortunately, biocompatibility is not measurable and
a clear operative definition of it does not exist. However, some tests and proce-
dures have been development to evaluate biocompatibility. Commonly, in vitro
tests are firstly carried out, followed by in vivo animal tests and clinical trials.
According to the particular application for which the device has been designed,
different kinds of tests can be performed, such as cytotoxicity, mutagenesis,
emocompatibility, etc.
These general considerations must be applied also to new ES materials aiming
at supporting cell growth for inducing tissue regeneration. In the course of this
Ph.D. the collaboration with biological laboratories enabled to carry out in vitro
tests to evaluate biocompatibility of newly synthesized polymers and copolymers.
As an example, biocompatibility studies of ES poly(x-pentadecalactone) scaffolds
will be presented in this paragraph. PPDL is a non-commercial polymer which is
synthesized at present only at laboratory scale. The PPDL used in the course of the
present research activity was synthesized by enzyme catalyzed polymerization and
it was ES by using organic solvents (CLF, DCM and HFIP, see Sect. 3.2.2) which
are highly toxic towards cells. Despite the fact that TGA analysis confirmed the
absence of detectable residual solvents, it is important to asses the effect of solvent
traces eventually contained in the ES fibres. The biocompatibility of PPDL ES
scaffolds towards mammalian cells was evaluated by using the H9c2 cell line
3.4 Cell Culture Experiments 101

(myoblast cells derived from embryonic rat heart). These cells were used as in
vitro benchmark to test indirect cytotoxicity as well as cell adhesion, proliferation
and morphology of cells seeded on PPDL fibres7 [143].
Indirect cytotoxicity was firstly investigated. This kind of test evaluates harmful
effects on mammalian cell cultures arising from the release of toxic substances
from the device. Indeed, all materials can release, to a different extent, low
molecular weight substances such as residual monomers, additives, impurities or
residual processing solvents (in the case of ES materials). Indirect cytotoxicity
evaluation of ES PPDL scaffolds was performed in accordance with the ISO10993-
5 international standard for biological evaluation of medical devices.8 In brief, ES
PPDL was kept in Dulbecco’s modified Eagle’s Medium (DMEM) in order to
extract the low molecular weight substances from the fibres. The DMEM con-
taining the PPDL extracted substances was added to a culture of H9c2 cells and
viable cells were quantified by using the sulforhodamine B (SRB) colorimetric
assay for cytotoxicity screening.9 The result was compared both with the negative
control (cells culture in DMEM not kept in contact with ES PPDL) and with the
positive cytotoxic control (cells culture in DMEM added with 1 mM H2O2).
Figure 3.51 shows that SRB absorption spectroscopy output was comparable for
samples grown for 24 h in PPDL-extraction medium or in standard DMEM
medium whereas, when exposed to 1 mM H2O2, as a positive cytotoxicity control,
all the cells died (not shown). This result indicates the absence of potentially
cytotoxic products released from PPDL, in agreement with the conclusion of
recent indirect cytotoxicity tests [144] run on mouse fibroblast 3T3 cells treated
with an extract from a PPDL bulk sample. In addition, we demonstrated that PPDL
non-cytotoxicity was maintained also after scaffold fabrication via electrospinning
by using CLF, DCM and HFIP as solvents.
Cytocompatibility tests were also carried out by seeding cells directly on PPDL
ES scaffolds.10 In order to evaluate cell adhesion and proliferation on the elec-
trospun scaffold, the number of viable cells was quantified every other day, up to

7
A PPDL solution (7% w/V) in CLF/DCM/HFIP (50/40/10, by volume) was electrospun using
the following process conditions: DV = 16 kV, R = 0.05 ml/min, d = 15 cm, T = 23 ± 2 C,
RH = 40 ± 5%.
8
PPDL mats were immersed in DMEM (5 mg polymer/1 ml medium) supplemented with 10%
heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 100 U/ml pen/strep, at 37 C
in a humidified atmosphere containing 5% CO2 for 24 h in order to obtain the medium containing
the PPDL (PPDL-extract medium). H9c2 cells were seeded in a 96-well culture plate (500 cells/
well) in standard DMEM to allow their attachment. After 48 h, the culture medium was
discarded, the PPDL-extract medium was added to the wells and the cells were further incubated
for 24 h.
9
Optical density (k = 540 nm) of samples was read in a Wallac VICTOR multilabel multiplate
reader (Perkin Elmer). Two separate experiments, six replicates each, were performed. Optical
density mean values ± standard error of the mean (sem) for replicates were calculated and the
unpaired t-test was used to evaluate statistical differences between mean values.
10
2.5 9 104 H9c2 cells in 1 ml DMEM were seeded onto the PPDL mat.
102 3 Results and Discussion

Fig. 3.51 Evaluation of the

indirect cytotoxicity of ES
PPDL scaffolds. Cell viability
in PPDL extraction media
was not statistically different
than that in DMEM
(Reprinted from [143],
Copyright (2010), with
permission from Brill
Publisher)

14 days, with the Alamar Blue fluorescence assay.11 The same cell culture was
monitored over time, thus overcoming drawbacks inherent to other sample-
destructive proliferation assays, such as the MTT and MTS tests. Control signal
was acquired from H9c2 cells cultured in TCPS. Results reported in Fig. 3.52
show that, after 24 h from cell seeding, ES PPDL mats hosted about 50% of
the number of H9c2 cells that adhered to the control polystyrene surface (day 1).
The number of cells growing onto PPDL increased linearly for up to day 14 (end of
experiments), although a significant difference with respect to the number of the
cells proliferating onto the TCPS was maintained at any given time point
(p \ 0.0001 by ANOVA). Alamar Blue assay showed that PPDL fibrous substrates
are non cytotoxic towards H9c2 cells and support cell proliferation. Moreover,
when let to grow up to 4 weeks, H9c2 cells reached the same maximum value
obtained on the polystyrene support (data not shown).
The morphology of cells grown onto ES PPDL mats was observed by SEM.
Figure 3.53a shows that, after 14 days of culture, H9c2 cells spread over the PPDL
mat surface while retaining their native, mesenchymal spindle-shaped, sheet-like
morphology. Furthermore, at 14 days, the scaffold surface was almost entirely
covered by cells. In the experiment where cells were allowed to grow over PPDL
for up to 4 weeks, the PPDL surface appeared completely covered with a cell
monolayer that prevented visualization of underlying fibres (Fig. 3.53b).
SEM observation of cell morphology together with results provided by indirect
cytotoxicity test and Alamar Blue assay confirmed that PPDL in the form of ES
fibres is biocompatible and is able to promote H9c2 adhesion and proliferation,
thus being a promising slow-degrading support for TE applications.

11
Blue fluorescence (Ex/Em = 540/590 nm) was read in a Wallac Victor multilabel multiplate
reader (Perkin Elmer). Four separate experiments (n = 4), three replicates each, were performed.
Two-way analysis of variance (ANOVA) was performed to compare proliferation curves. Values
were given as the mean values of fluorescence ± sem.
3.4 Cell Culture Experiments 103

3.4.4 Electrospun Fibres Loaded with Growth Factor: Effect

on Stem Cell Culture

GFs are proteins dissolved in the gel-like component of ECM and have a key role
in driving cell activity. They are frequently added to cell culture media during
common in vitro cell culture experiments in order to induce cell proliferation and
differentiation. Given their biological importance, several efforts are made in TE
for achieving a gradually release of GFs from the scaffold upon contact with
physiological fluids.
In the course of this Ph.D. an Endothelial Cell Growth Factor Supplement
(ECGS) from Bovine Neural Tissue (Sigma–Aldrich) was incorporated within ES
fibres through a one-step process. PLA50GA50 ES scaffolds loaded with different
amounts of ECGS were prepared as previously described in Sect. 3.2.6.1. ECGS is
typically formulated to stimulate cell proliferation activity and to induce stem cell

Fig. 3.52 Evaluation of cell

adhesion and proliferation on
the ES PPDL scaffolds
(continuous line) compared
with TCPS control (dotted
line) by Alamar Blue
fluorescence assay (Reprinted
from [143], Copyright (2010),
with permission from Brill
Publisher)

Fig. 3.53 SEM micrographs showing the interaction between H9c2 cells and PPDL ES scaffold
after 14 days (a) and 27 days of culture (b) (Reprinted from [143], Copyright (2010), with
permission from Brill Publisher)
104 3 Results and Discussion

differentiation towards endothelial lineage. ES mats supplemented with ECGS

where exposed to a culture of Mesenchymal Cells, derived from human bone
marrow,12 with the aim to evaluate the effect of ECGS on cell behavior. The final
goal was to assess if ECGS retains its bioactivity once released in the culture
medium from the ES fibres [97].
MSCs were cultured13 in the presence of both plain scaffolds (0-ECGS) and of
ECGS-loaded scaffolds (0.4-ECGS and 4.8-ECGS, where the number indicates the
ECGS content in wt%). In the course of this experiment cells were not seeded on
the ES scaffold but on 2D TCPS, while the mat floated in the culture medium. This
procedure allowed to investigate the influence of the GF released from the fibres
on cell growth, without introducing any additional effects on cell culture, such as
the nature of a 3D substrate. Control experiments were also run without ES mat in
the culture dish: as a positive control, cell culture was supplemented with ECGS in
a concentration routinely used in cell culture (100 lg/ml); as a negative control an
identical experiment was run without ECGS addition. Cell proliferation, cell
spreading and shape were evaluated after 7 days of culture.
Cell proliferation was evaluated: (1) through the estimation of the percentage of
cells expressing the intranuclear protein Ki-6714 [145] and (2) by directly counting
cell number on images acquired by optical microscopy.15 Figure 3.54 shows that the
percentage of cells expressing the Ki-67 after 7 days of culture was higher in both the
positive control and in the 0.4-ECGS and 4.8-ECGS experiments when compared to
the percentage of Ki-67 positive cells seen in negative control and 0-ECGS. This
result indicates that cells were stimulated to proliferate when cultured in the presence
of directly supplemented ECGS, as well as of ECGS-loaded scaffolds.

12
The bone marrow collected from a healthy donor was diluted in culture medium (Medium
199, Sigma–Aldrich) supplemented with 0.5% Fetal Calf Serum (FCS, Sigma–Aldrich) and
1000 U/ml heparin. The diluted bone marrow was layered over a Ficoll-Paque solution and
centrifuged at 1100 RPM for 30 min. The cells were washed twice in culture medium, suspended
in Medium 199 supplemented with 10% FCS and seeded in 25 cm2 culture dishes. After 24 h the
non adherent cells were removed, while the adherent cells were cultured at 37 C in 5% CO2
atmosphere. The culture medium was changed every 2–3 days. After a week a cell monolayer
was achieved.
13
Approximately 1 9 106 cells were seeded in 25 cm2 TCPS culture dishes in 15 ml of Medium
199 (Sigma–Aldrich) supplemented with 10% Fetal Calf Serum (FCS, from Sigma) for 7 days.
The medium was changed every 2–3 days.
14
Cells were detached from TCPS using 0.25% Trypsin/EDTA solution and the intranuclear Ki-
67 protein was estimated by flow cytometry (Beckman Coulter FC 500 Flow Cytometer equipped
with argon laser, k = 488 nm) by using a Fluorescein FITC conjugated monoclonal antibody
(Becton–Dickinson & Co Industry). Values were given as mean percentage cells positive to Ki-
67 ± standard error of the mean (sem) and the unpaired t-test was used to evaluate statistical
differences between mean values.
15
Images of the cells were taken at the beginning of the experiments (day 0) and at the end of
the 7th day. Cell counting was performed using an AxioObserver microscope (Zeiss) by means of
the image analysis software AxioVision 4.6 (Zeiss). Cell number was measured on
350 lm 9 350 lm squared areas and the results obtained from 4 different areas per sample
were averaged. Values were given as mean ± standard deviation.
3.4 Cell Culture Experiments 105

Fig. 3.54 Percentage of

MSCs cells expressing Ki67
after 7 days of culture
(Reprinted from [97],
Copyright (2009), with
permission from e-polymers)

Table 3.6 Cell number changes after 7 days of culture

Experiment Cell numbera Cell number increment (%)
Day 0 Day 7
Negative controlb 79 (9) 86 (4) + 9
Positive controlc 81 (8) 115 (3) + 42
0-ECGSd 54 (4) 59 (2) + 9
0.4-ECGSd 67 (2) 97 (4) + 45
4.8-ECGSd 54 (3) 85 (2) + 57
Reprinted from [97], Copyright (2009), with permission from e-polymers
a
From Differential Interference Contrast Microscopy, standard deviation in parenthesis
b
Cell culture without ECGS addition
c
Cell culture with ECGS addition (100 lg/ml)
d
Cell culture containing the indicated ES fibre mat

Cell proliferation was also estimated by counting the number of cells attached
to the TCPS at the beginning of the experiment (day 0) and after 7 days of culture.
Cell density varied from sample to sample so cell proliferation was estimated by
calculating the percentage increment of cell number. Table 3.6 lists cell number at
day 0 and at day 7 as well as the observed percentage increment. It is clear that
both negative control and culture containing the scaffold without ECGS (0-ECGS)
underwent a cell increment of less than 10%, whereas the cell increment reached
around 50% in all other experiments (positive control, 0.4-ECGS, 4.8-ECGS). This
result agrees with the trend of the percentage of cells expressing Ki-67 discussed
above (Fig. 3.54).
Cell spreading and cell shape, which are known to be strong determinants of the
cell fate [146, 147], were investigated by direct observation of cell culture images
acquired by DIC microscopy. Figure 3.55 reports some representative images of
cell cultured in the presence of ES meshes (0-ECGS, 0.4-ECGS and 4.8-ECGS)
together with negative and positive controls. It is clearly observed that the exposure
to ECGS (either directly supplemented in the positive control or released from the
ECGS-loaded scaffolds) not only promoted cell growth but also affected cell area
106 3 Results and Discussion

Fig. 3.55 Comparison of

DIC micrographs at day 0 and
day 7, in different culture
conditions. Scale
bar = 20 lm (Reprinted
from [97], Copyright (2009),
with permission from
e-polymers)

and overall cell shape. By measuring cell area (A) and cell perimeter (P)16 the cell
shape index, that is directly correlated to cell fate, was calculated as follows:
4p A
S¼ ð3:2Þ
P2
The shape index of an ideal circle is 0, while that of a linear object is 1.
Table 3.7 reports both cell areas and cell shape indexes calculated for all the
cell culture conditions at day 0 and at day 7. The shape index of the cells in the
negative control and that of the cells cultured in the presence of 0-ECGS slightly
increased after 7 days. On the contrary, the shape index of cells from the cultures
containing ECGS-loaded mats decreased after 7 days from 0.61 to 0.49 and from
0.74 to 0.41, in 0.4-ECGS and 4.8-ECGS respectively. These values were

16
Area and perimeter were individually estimated for each cell contained in the four analyzed
350 lm 9 350 lm TCPS portions, and the average A and P values were used in Eq. 3.2.
3.4 Cell Culture Experiments 107

Table 3.7 Cell morphology changes after 7 days of cell culture

Experiment Shape indexa Cell area (lm2)b
Day 0 Day 7 Day 0 Day 7
Negative controlc 0.45 (0.04) 0.64 (0.02) 346 (8) 352 (6)
Positive controld 0.67 (0.03) 0.41 (0.03) 342 (3) 416 (5)
0-ECGSe 0.56 (0.05) 0.59 (0.02) 343 (3) 366 (3)
0.4-ECGSe 0.61 (0.03) 0.49 (0.03) 392 (4) 474 (7)
4.8-ECGSe 0.74 (0.03) 0.41 (0.02) 354 (4) 401 (2)
Reprinted from [97], Copyright (2009), with permission from e-polymers
a
Calculated according to Eq. 3.2, standard deviation in parenthesis
b
Measured for each cell contained in four analyzed 350 lm 9 350 lm cell TCPS portions,
standard deviation in parenthesis
c
Cell culture without ECGS addition
d
Cell culture with ECGS addition (100 lg/ml)
e
Cell culture containing the indicated ES fibre mat

comparable to those seen in the positive control where the shape index also
decreased from 0.67 (day 0) to 0.41 (day 7). The shape index can assume two limit
values, namely 0 and 1, corresponding to circular and linear shape respectively.
Hence, the decrease of shape index observed in the ECGS-supplemented culture
and in cultures containing ECGS-loaded mats seems to indicate a tendency of the
cells to adopt non-elongated shapes.
Visual inspection of the pictures in Fig. 3.55 shows that at day 0 most of the cells
had round or elongated shape. At day 7, in the positive control and in the cultures
with 0.4-ECGS and 4.8-ECGS a rather clear change in cell shape could be appre-
ciated. Many cells assumed a romboidal shape, in agreement with the mentioned
shape index change. In culture, mesenchymal cells can assume distinct yet subtle
shape differences in relation to their phenotype lineage; for instance, the shape index
of endothelial-like cells is expected to be smaller (in the range 0–0.5) than that of
fibroblast-like cells (range 0.5–1). Whether the observed cell shape change could
correspond to a true phenotypic shift toward an endothelial-like lineage remains to
be established. Taken together these data indicate that ECGS delivered from the
scaffolds retains an activity comparable to that of the positive control. ES metho-
dology is therefore suitable for producing bioactive nanofibrous scaffolds through
the incorporation of GFs than can be released without loss of bioactivity.

3.4.5 Effect of Electrospun Fibre Orientation on Cancer Cell

Culture

In the course of the present Ph.D. ES materials have been designed and fabricated
to pursuit the scopes of tissue engineering. This final section illustrates an alter-
native application of ES materials which, similarly to TE, will probably have a
great impact in the future of life sciences. A relatively new application of 3D
scaffolds concerns the development of new in vitro models for the study of disease
108 3 Results and Discussion

pathogenesis [148, 149]. Indeed, essential cellular functions, that are normally
displayed in tissues, are not observable when cells are cultured in TCPS plates
(i.e. on a flat 2D surface). Therefore, to date, serious limitations in predicting the
cellular responses in the organisms exist. As previously discussed in Chap. 1,
when compared to 2D TCPS in vitro cultures, the phenotype assumed by cells on
3D substrates better resembles that developed in vivo. Therefore, the use of 3D
scaffolds as new in vitro models is expected to bridge the gap between 2D flat cell
culture and living tissues and, consequently, enormous progress in the field of drug
screening and reduction in the use of animal models may be foreseen [150].
One of the fields that is currently under investigation is the use of scaffolds as
3D in vitro cell culture models to improve tumour modelling [151]. In vitro cancer
models are valuable systems for studying cell behaviour under controlled condi-
tions and under specific therapeutic treatments for circumventing the complexity
of in vivo systems. However, researches in cancer pathologies are limited by the
experimental instruments currently available for studying these complex diseases.
Cancer cells, which are abnormal cells displaying an uncontrolled growth, have the
ability to invade surrounding tissues with a large variety of migration modalities.
The hallmark of cancer malignity in vivo is invasion. Tumour cells mainly migrate
in form of aggregates that assume different shapes [152]. For the sake of sim-
plicity, two main types of cell aggregation can be identified: (1) nest aggregates
and (2) chain aggregates. The latter migration mode is commonly associated with
tumours exhibiting high level of malignity, because cell penetration into the sur-
rounding tissues is extremely effective. To date it is not clear yet which are the
chemical and/or the morphological signals that address cells to a specific migration
mode.
With the aim to reproduce an in vitro environment resembling the in vivo ECM
organization, ES scaffolds were used as 3D porous substrates to culture mam-
malian cancer cells. ES mats composed of differently oriented P(L)LA fibres (both
randomly arranged and highly aligned) were produced.17 MCF7 cells derived from
a mammary carcinoma cell line18 were used and cell culture on the ES scaffolds
was carried out for 7 days.19 This experiment aims at investigating the effect of

17
A P(L)LA solution (13% w/V) in DCM/DMF (65:35, by volume) was electrospun using the
following process conditions: DV = 12 kV, R = 0.015 ml/min, d = 15 cm, T = 22 ± 1 C,
RH = 35 ± 3%. Meshes with randomly arranged fibres were obtained by collecting the fibres on
a cylindrical target (radius = 25 mm) rotating at angular rate x = 200 rpm. Meshes made of
highly aligned fibres were obtain by collecting the fibres on the same cylindrical target rotating at
angular rate x = 6200 rpm.
18
Human breast cancer cell line MCF7 were maintained in RPMI-1640 medium supplemented
with 10% fetal bovine serum (FBS), 100 lg/ml Streptomycin, 100 IU/ml Penicillin, 2 mM
L-Glutamine and 0.1 mM non-essential amino acids in T-75 flasks in an incubator at 37 C
and 5% CO2. Sub-confluence cell were trypsinized (0.05% Trysin-EDTA), centrifuged and
re-suspended in culture medium.
19
1 9 105 breast cancer cells MCF7 were seeded in 1 mL of complete RPMI-1640 medium
onto the PLLA ES scaffolds and cultured in standard culture conditions (37 C, 5% CO2) for
7 days.
3.4 Cell Culture Experiments 109

Fig. 3.56 Evaluation of cell

adhesion and proliferation on
both ES P(L)LA random and
aligned fibres compared with
polystyrene control by
Alamar Blue fluorescence
assay

fibre spatial arrangement on cancer cell behaviour in order to better understand the
role of the fibrous component of ECM in driving the development of tumour forms
in the organism.
Cell adhesion and proliferation on mats composed of differently oriented fibres
were evaluated by Alamar Blue assay and results were compared to culture
experiment performed on TCPS as a control (Fig. 3.56). The number of viable
cells was quantified every other day, up to 7 days. After 24 h from cell seeding,
both ES P(L)LA mats (i.e. mat composed of randomly oriented fibres and mat
composed of aligned fibres) host about 50% of the number of cells that adhere to
the control TCPS surface (day 1). The number of cells growing onto both P(L)LA
mats slightly increased in the following days, although a significant difference with
respect to the number of the cells proliferating onto the TCPS is maintained at any
given time point (p \ 0.0001 by ANOVA). Alamar Blue assays showed that
P(L)LA fibres are able to host MCF7 cells and that cell proliferation is not affected
by scaffold morphology.
Interestingly and contrary to cell proliferation, cell morphology appeared to be
highly dependent on the geometry of the substrate. Figure 3.57 shows SEM
micrographs of MCF7 after 7 days of culture on P(L)LA fibres differently oriented
(both random and aligned fibres) at different magnifications. Cancer cells aggre-
gated on both types of substrates. On random fibres cells generated nest aggregates
(Fig. 3.57a, c and e, different magnifications) whereas on aligned fibres both nests
(Fig. 3.57d, black circles) and chains (Fig. 3.57d, white circles) could be observed.
Higher magnification micrographs clearly shows the different aggregate mor-
phologies assumed by cancer cells on random fibres (Fig. 3.57e) and on aligned
fibres (Fig. 3.57f, where a detail of aggregated cells in single row is shown). It is
worth noting that, on aligned fibres, both chain aggregates and single cells (white
arrows in Fig. 3.57f) are oriented along fibre direction. The tendency of a cell to
orient its cytoplasm towards fibre direction when cultured on aligned fibrous
substrates has been observed for several cell types [39, 153–156] while the
110 3 Results and Discussion

Fig. 3.57 SEM micrographs of MCF7 after 7 days of culture on P(L)LA differently oriented
fibres. Cancer cells generated nest aggregate when cultured on random fibres (a, c and e, different
magnifications) whereas they generated both nests (dark circles) and chains (white circles) when
cultured on aligned fibres (b, d and f, different magnifications)

cooperative orientation of cells in order to generate an oriented aggregate has not

been documented yet.
These preliminary results seem to indicate a strong influence of substrate
geometry on cancer cell collective behaviour. Since ES fibrous scaffolds mimic the
organization of ECM fibrous component, this finding might be a key information
in better understanding the role of natural ECM on tumour malignity in vivo.
Moreover, it is pointed out that, if nest aggregates are easily observed on 2D TCPS
in vitro cultures, chain aggregates cannot be reproduced in vitro by using the
currently available TCPS flat substrates. On the contrary, the use of 3D ES scaf-
folds helps reproducing the morphologies that MCF7 cells can assume in vivo,
3.4 Cell Culture Experiments 111

demonstrating to be a powerful substrate for modelling cancer cell response in

vitro. However, results provided by Alamar Blue assays and SEM observations are
far from being exhaustive. A deeper investigation of cancer cell behaviour on 3D
ES scaffolds will be carried out in the next future.

References

1. Eichhorn SJ, Sampson WW (2005) Statistical gometry of pores and statistics of porous
nanofibrous assemblies. J R Soc Interface 2:309
2. Barry JJA, Gidda HS, Scotchford CA, Howdle SM (2004) Porous methacrylate scaffolds:
supercritical fluid fabrication and in vitro chondrocyte responses. Biomaterials 25:3559
3. Barry JJA, Silva MCG, Cartmell SH, Guldberg RE, Scotchford CA, Howdle SM (2006)
Porous methacrylate tissue engineering scaffolds: using carbon dioxide to control porosity
and interconnectivity. J Mater Sci 41:4197
4. Barry JJA, Nazhat SN, Rose FRAJ, Hainsworth AH, Scotchford CA, Howdle SM (2005)
Supercritical carbon dioxide foaming of elastomer/heterocyclic methacrylate blends as
scaffolds for tissue engineering. J Mater Chem 15:4881
5. Tai H, Mather ML, Howard D, Wang W, White LJ, Crowe JA, Morgan SP, Williams DJ,
Howdle SM, Shakesheff KM (2007) Control of pore size and structure of tissue engineering
scaffolds produced by supercritical fluid processing. Eur Cell Mater 14:64
6. Lin ASP, Barrows TH, Cartmell SH, Guldberg RE (2003) Microarchitectural and
mechanical characterization of oriented porous polymer scaffolds. Biomaterials 24:481
7. Howdle SM, Watson MS, Whitaker MJ, Popov VK, Davies MC, Mandel FS, Don Wang J,
Shakesheff KM (2001) Supercritical fluid mixing: preparation of thermally sensitive
polymer composites containing bioactive materials. Chem Commun 1:109
8. Mather ML, Brion M, White LJ, Shakesheff KM, Howdle SM, Morgan SP, Crowe JA
(2009) Time-lapsed imaging for in-process evaluation of supercritical fluid processing of
tissue engineering scaffolds. Biotechnol Prog 25:1176
9. Singh L, Kumar V, Ratner BD (2004) Generation of porous microcellular 85/15 poly
(dl-lactide-co-glycolide) foams for biomedical applications. Biomaterials 25:2611
10. Arora KA, Lesser AJ, McCarthy TJ (1998) Preparation and characterization of microcellular
polystyrene foams processed in supercritical carbon dioxide. Macromolecules 31:4614
11. Goel SK, Beckman EJ (1994) Generation of microcellular polymeric foams using
supercritical carbon dioxide. I: effect of pressure and temperature on nucleation. Polym
Eng Sci 34:1137
12. Mooney DJ, Baldwin DF, Suh NP, Vacanti JP, Langer R (1996) Novel approach to fabricate
porous sponges of poly(D, L-lactic-co-glycolic acid) without the use of organic solvents.
Biomaterials 17:1417
13. Davies OR, Lewis AL, Whitaker MJ, Tai H, Shakesheff KM, Howdle SM (2008)
Applications of supercritical CO2 in the fabrication of polymer systems for drug delivery
and tissue engineering. Adv Drug Deliv Rev 60:373
14. Oliveira NS, Dorgan J, Coutinho JAP, Ferreira A, Daridon JL, Marrucho IM (2006) Gas
solubility of carbon dioxide in poly(lactic acid) at high pressures. J Polym Sci Part B Polym
Phys 44:1010
15. Gualandi C, White LJ, Chen L, Gross RA, Shakesheff KM, Howdle SM, Scandola M (2010)
Scaffold for tissue engineering fabricated by non-isothermal supercritical carbon dioxide
foaming of a highly crystalline polyester. Acta Biomater 6:130
16. Ceccorulli G, Scandola M, Kumar A, Kalra B, Gross RA (2005) Cocrystallization of
random copolymers of x-pentadecalactone and e-caprolactone synthesized by lipase
catalysis. Biomacromolecules 6:902
112 3 Results and Discussion

17. Doroudiani S, Park CB, Kortschot MT (1996) Eflect of the crystallinity and morphology on
the microcellular foam structure of semicrystalline polymers. Polym Eng Sci 36:2645
18. Tomasko DL, Li H, Liu D, Han X, Wingert MJ, Lee LJ, Koelling KW (2003) A review of
CO2 applications in the processing of polymers. Ind Eng Chem Res 42:6431
19. Mathieu L, Montjovent M-O, Bourban P-E, Pioletti DP, Manson J-AE (2005) Bioresorbable
composites prepared by supercritical fluid foaming. J Biomed Mater Res Part A 75:89
20. Fox TG (1956) Influence of diluent and of copolymer composition on the glass temperature
of a polymer system. Bull Am Phys Soc 1:123
21. Couchman PR, Karasz FE (1978) A classical thermodynamic discussion of the effect of
composition on glass-transition temperatures. Macromolecules 11:117
22. Chow TS (1980) Molecular interpretation of the glass transition temperature of polymer-
diluent systems. Macromolecules 13:362
23. Flory PJ (1953) Principles of polymer chemistry. Cornell University Press, New York
24. Gibson LJ (2005) Biomechanics of cellular solids. J Biomech 38:377
25. Black J, Hastings G (1998) Handbook of biomaterial properties. Chapman & hall, London
26. Kutz M (2003) Standard handbook of biomedical engineering and design. The McGraw-
Hall Companies, New York
27. Fridrikh SV, Yu JH, Brenner MP, Rutledge G (2003) Controlling the fiber diameter during
electrospinning. Phys Rev Lett 90:144502-1
28. Shin M, Hohman MM, Brenner MP, Rutledge G (2001) Electrospinning: a whipping fluid
jet generates submicron polymer fibers. Appl Phys Lett 78:1149
29. Helgeson ME, Grammatikos KN, Deitzel JM, Wagner NJ (2008) Theory and kinematic
measurement of the mechanics of stable electrospun polymer jets. Polymer 49:2924
30. Reneker DH, Yarin AL, Fong H, Koombhongse S (2000) Bending instability of electricaly
charged liquid jets of polymer solutions in electrospinning. J Appl Phys 87:4531
31. Shin YM, Hohman MM, Brenner MP, Rutledge GC (2001) Experimental characterization
of electrospinning: the electrically forced jet and instabilities. Polymer 42:9955
32. Reneker DH, Yarin AL, Zussman E, Koombhongse S, Kataphinan W (2006) Nanofiber
manufacturing: toward better process control. In: Reneker DH, Fong H (eds) Polymeric
Nanofibers, ACS Symposium Series 918. Oxford University Press, Oxford, pp 7–20
33. Taylor G (1969) Electrically driven jets. Proc R Soc Lond 313:453
34. Lee JS, Choi KH, Ghim HD, Kim S-S, Chun DH, Kim HY, Ma K (2004) Role of molecular
weight of atactic poly(vinyl alcohol) (PVA) in the structure and properties of PVA
nanofabric prepared by electrospinning. J Appl Polym Sci 93:1638
35. Tan S-H, Inai R, Kotaki M, Ramakrishna S (2005) Systematic parameter study for ultra-fine
fiber fabrication via electrospinning process. Polymer 46:6128
36. Katti DS, Robinson KW, Ko FK, Laurencin CT (2004) Bioresorbable nanofiber-based
systems for wound healing and drug delivery: optimization of fabrication parameters.
J Biomed Mater Res B Appl Biomater 70B:286
37. Megelski S, Stephens JS, Chase DB, Rabolt JF (2002) Micro- and nanostructured surface
morphology on electrospun polymer fibers. Macromolecules 35:8456
38. Kidoaki S, Kwon IK, Matsuda T (2006) Structural features and mechanical properties of in
situ–bonded meshes of segmented polyurethane electrospun from mixed solvents. J Biomed
Mater Res B Appl Biomater 76B:219
39. Baker SC, Atkin N, Gunning PA, Granville N, Wilson K, Wilson D, Southgate J (2006)
Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro
biological studies. Biomaterials 27:3136
40. Gupta P, Elkins C, Long TE, Wilkes GL (2005) Electrospinning of linear homopolymers of
poly(methyl methacrylate): exploring relationships between fiber formation, viscosity,
molecular weight and concentration in a good solvent. Polymer 46:4799
41. Shenoy SL, Bates WD, Frisch HL, Wnek GE (2005) Role of chain entanglements on fiber
formation during electrospinning of polymer solutions: good solvent, non-specific polymer-
polymer interaction limit. Polymer 46:3372
References 113

42. Jarusuwannapoom T, Hongrojjanawiwat W, Jitjaicham S, Wannatong L, Nithitanakul M,

Pattamaprom C, Koombhongse P, Rangkupan R, Supaphol P (2005) Effect of solvents on
electro-spinnability of polystyrene solutions and morphological appearence of resulting
electrospun polystyrene fibers. Eur Polym J 41:409
43. Son WK, Youk JH, Lee TS, Park WH (2004) The effects of solution properties and
polyelectrolyte on electrospinning of ultrafine poly(ethylene oxide) fibers. Polymer 45:2959
44. Lide DR (2007) Handbook of chemistry and physics. CRC Press, Boca Raton
45. Zhong XH, Kim K, Fang D, Ran S, Hsiao BS, Chu B (2002) Structure and process
relationship of electrospun bioabsorbable nanofiber membranes. Polymer 43:4403
46. Fong H, Reneker DH (1999) Beaded nanofibers formed during electrospinning. Polymer
40:4585
47. Ramakrishna S, Fujihara K, Teo W-E, Lim T, Ma Z (2005) An introduction to
electrospinning and nanofibers. World Scientific Publishing, Singapore
48. Lee KH, Kim HY, Bang HJ, Jung YH, Lee SG (2003) The change of bead morphology
formed on electrospun polystyrene fibers. Polymer 44:4029
49. Vert M, Schwarch G, Czerwonka LA (1995) Present and future of PLA polymers.
J Macromol Sci Chem 32:787
50. Middleton JC, Tipton AJ (2000) Synthetic biodegradable polymers as orthopedic devices.
Biomaterials 21:2335
51. Engelberg I, Kohn J (1991) Physico-mechanical properties of degradable polymers used in
medical applications: a comparative study. Biomaterials 12:292
52. Fischer EW, Sterzel HJ, Wegner G (1973) Investigation of the structure of solution grown
crystals of lactide copolymers by means of chemical reactions. Kolloid-Z u Z Polymere
251:980
53. Garlotta D (2001) A literature review of poly(lactic acid). J Environ Polym Degr 9:63
54. Henton DE, Gruber PR, Lunt J, Randall J (2005) Polylactic acid technology. In: Mohanty AK,
Misra M, Drzal LT (eds) Natural Fibers, Biopolymers and Biocomposites. CRC Press, Boca
Raton, pp 527–578
55. Pego AP, Poot AA, Grijpma DW, Feijen J (2003) Biodegradable elastomeric scaffolds for
soft tissue engineering. J Controlled Release 87:69
56. Plikk P, Malberg S, Albertsson A-C (2009) Design of resorbable porous tubular copolyester
scaffolds for use in nerve regeneration. Biomacromolecules 10:1259
57. Geminiani G (2009) Studio di materiali polimerici nanofibrosi con proprietà elastomeriche
per usi biomedicali. Chemistry Dept ‘‘G. Ciamician’’, University of Bologna, Chemistry
Eurobachelor
58. Pitt CG (1990) Poly-e-caprolactone and its copolymers. In: Chasin M, Langer R (eds)
Biodegradable Polymers as Drug Delivery Systems. Marcel Dekker, New York, pp 71–119
59. Zhong X, Ran S, Fang D, Hsiao BS, Chu B (2003) Control of structure, morphology and
property in electrospun poly(glycolide-co-lactide) non-woven membranes via post-draw
treatments. Polymer 44:4959
60. Deitzel JM, Kleinmeyer JD, Hirvonen JK, Beck Tan NC (2001) Controlled deposition of
electrospun poly(ethylene oxide) fibers. Polymer 42:8163
61. Pitt CG, Chasalow FI, Hibionada YM, Klimas DM (1981) Aliphatic polyesters. I. The
degradation of poly(e-caprolactone) in vivo. J Appl Polym Sci 26:3779
62. Focarete ML, Gazzano M, Scandola M, Gross RA (2002) Copolymers of x-pentadecalactone
and trimethylene carbonate from lipase catalysis: influence of microstructure on solid-state
properties. Macromolecules 35:8066
63. Jiang Z, Azim H, Gross RA, Focarete ML, Scandola M (2007) Lipase-catalyzed copoly-
merization of x-pentadecalactone with p-dioxanone and characterization of copolymer thermal
and crystalline properties. Biomacromolecules 8:2262
64. Shawon J, Sung C (2004) Electrospinning of polycarbonate nanofibers with solvent
mixtures THF and DMF. J Mater Sci 39:4605
114 3 Results and Discussion

65. Casper CL, Stephens JS, Tassi NG, Chase DB, Rabolt JF (2004) Controlling surface
morphology of electrospun polystyrene fibers: effect of humidity and molecular weight in
electrospinning process. Macromolecules 37:573
66. Teo W-E, Ramakrishna S (2006) A review on electrospinning design and nanofibre
assemblies. Nanotechnology 17:R89
67. Matthews JA, Wnek GE, Simpson DG, Bowlin GL (2002) Electrospinning of collagen
nanofibers. Biomacromolecules 3:232
68. Katta P, Alessandro M, Ramsier RD, Chase GG (2004) Continuous electrospinning of
aligned polymer nanofibers onto a wire drum collector. Nano Lett 4:2215
69. Wu Y, Carnell LA, Clark RL (2007) Control of electrospun mat width through the use of
parallel auxiliary electrodes. Polymer 48:5653
70. Carnell LS, Siochi EJ, Holloway NM, Stephens RM, Rhim C, Niklason LE, Clark RL
(2008) Aligned mats from electrospun single fibers. Macromolecules 41:5345
71. Teo W-E, Kotaki M, Mo XM, Ramakrishna S (2005) Porous tubular structures with
controlled fibre orientation using a modified electrospinning method. Nanotechnology
16:918
72. Li D, Wang YL, Xia YN (2004) Electrospinning nanofibers as uniaxially aligned arrays and
layer-by-layer stacked films. Adv Mat 16:361
73. Liu L, Dzenis Y (2008) Analysis of the effects of the residual charge and gap size on
electrospun nanofiber alignment in a gap method. Nanotechnology 19:355307
74. Li D, Ouyang G, McCann JT, Xia Y (2005) Collecting electrospun nanofibers with
patterned electrodes. Nano Lett 5:913
75. Zhang D, Chang J (2007) Patterning of electrospun fibers using electroconductive
templates. Adv Mater 19:3664
76. Gibson P, Schreuder-Gibson H (2004) Patterned electrospray fiber structures. Int
Nonwovens J 13:34
77. Ramakrishna S, Fujihara K, Teo W-E, Lim T, Ma Z (2005) Electrospinning process.
In: Ramakrishna S, Fujihara K, Teo W-E, Lim T, Ma Z (eds) An Introduction to
Electrospinning and Nanofibers. World Scientific Publishing, Singapore, pp 135–137
78. Zucchelli A, Fabiani D, Gualandi C, Focarete ML (2009) An innovative and versatile
approach to design highly porous, micropatterned, nanofibrous polymeric materials. J Mater
Sci 44:4969
79. Vargin VV (1967) Technology of enamels. MacLaren and Sons Ltd, London
80. Tavgen VV (2000) Glass ceramic enamels with increased conductivity. Glass Ceram 57:183
81. Kidoaki S, Kwon IK, Matsuda T (2005) Mesoscopic spatial designs of nano- and microfiber
meshes for tissue-engineering matrix and scaffold based on newly devised multilayering and
mixing electrospinning techniques. Biomaterials 26:37
82. Sala F (2009) Studio delle proprietà meccaniche di supporti polimerici elettrofilati
e sviluppo di scaffold tubolari per la rigenerazione del tessuto nervoso. Chemistry Dept
‘‘G. Ciamician’’, University of Bologna, Chemistry Eurobachelor
83. Hong Y, Fujimoto K, Hashizume R, Guan J, Stankus JJ, Tobita K, Wagner WR (2008)
Generating elastic, biodegradable polyurethane/poly(lactide-co-glycolide) fibrous sheets
with controlled antibiotic release via two-stream electrospinning. Biomacromolecules
9:1200
84. Huang Z-M, He CL, Yang A, Zhang Y, Han X-J, Yin J (2006) Encapsulating drugs in
biodegradable ultrafine fibers through co-axial electrospinning. J Biomed Mater Res Part A
77A:169
85. Kim K, Luu YK, Chang C, Fang D, Hsiao BS, Chu B, Hadjiargyrou M (2004) Incorporation
and controlled release of a hydrophilic antibiotic using poly(lactide-co-glycolide)-based
electrospun nanofibrous scaffolds. J Controlled Release 98:47
86. Kenawy ER, Abdel-Hay FI, El-Newehy MH, Wnek GE (2009) Processing of polymer
nanofibers through electrospinning as drug delivery systems. Mater Chem Phys 113:296
References 115

87. Piras AM, Nikkola L, Chiellini F, Ashammakhi N, Chiellini E (2006) Development

of diclofenac sodium releasing bio-erodible polymeric nanomats. J Nanosci Nanotechnol
6:3310
88. Taepaiboon P, Rungsardthong U, Supaphol P (2006) Drug-loaded electrospun mats of
poly(vinyl alcohol) fibres and their release characteristics of four model drugs.
Nanotechnology 17:2317
89. Xie J, Wang C-H (2006) Electrospun micro- and nanofibers for sustained delivery of
paclitaxel to treat C6 glioma in vitro. Pharm Res 23:1817
90. Casper CL, Yang WD, Farach-Carson MC, Rabolt JF (2007) Coating electrospun collagen
and gelatin fibers with perlecan domain I for increased growth factor binding.
Biomacromolecules 8:1116
91. Lu Y, Jiang HL, Tu KH, Wang LQ (2009) Mild immobilization of diverse macromolecular
bioactive agents onto multifunctional fibrous membranes prepared by coaxial electrospinning.
Acta Biomater 5:1562
92. Choi JS, Leong KW, Yoo HS (2008) In vivo wound healing of diabetic ulcers using
electrospun nanofibers immobilized with human epidermal growth factor (EGF). Biomaterials
29:587
93. Chew SY, Mi R, Hoke A, Leong KW (2007) Aligned protein-polymer composite fibers
enhance nerve regeneration: a potential tissue-engineering platform. Adv Funct Mater
17:1288
94. Chew SY, Wen J, Yim EKF, Leong KW (2005) Sustained release of proteins from
electrospun biodegradable fibers. Biomacromolecules 6:2017
95. Su Y, Li XQ, Tan LJ, Huang C, Mo XM (2009) Poly(L-lactide-co-e-caprolactone)
electrospun nanofibers for encapsulating and sustained releasing proteins. Polymer 50:4212
96. Liao IC, Chew SY, Leong KW (2006) Aligned core-shell nanofibers delivering bioactive
proteins. Nanomedicine 1:465
97. Gualandi C, Wilczek P, Focarete ML, Pasquinelli G, Kawalec M, Scandola M (2009)
Bioresorbable electrospun nanofibrous scaffolds loaded with bioactive molecules. e-Polymers
60
98. Yoo HS, Kim TG, Park TG (2009) Surface-functionalized electrospun nanofibers for tissue
engineering and drug delivery. Adv Drug Deliv Rev 61:1033
99. Zhu X, Chian KS, Chan-Park MBE, Lee ST (2005) Effect of argon-plasma treatment on
proliferation of human-skin-derived fibroblast on chitosan membrane in vitro. J Biomed
Mater Res Part A 73A:264
100. Venugopal J, Low S, Choon AT, Kumar AB, Ramakrishna S (2008) Electrospun-modified
nanofibrous scaffolds for the mineralization of osteoblast cells. J Biomed Mater Res Part A
85A:408
101. Prabhakaran MP, Venugopal J, Chan CK, Ramakrishna S (2008) Surface modified
electrospun nanofibrous scaffolds for nerve tissue engineering. Nanotechnology 19:455102
102. He W, Ma ZW, Yong T, Teo WE, Ramakrishna S (2005) Fabrication of collagen-coated
biodegradable polymer nanofiber mesh and its potential for endothelial cells growth.
Biomaterials 26:7606
103. Koh HS, Yong T, Chan CK, Ramakrishna S (2008) Enhancement of neurite outgrowth
using nano-structured scaffolds coupled with laminin. Biomaterials 29:3574
104. Choi WS, Bae JW, Lim HR, Joung YK, Park JC, Kwon IK, Park KD (2008) RGD peptide-
immobilized electrospun matrix of polyurethane for enhanced endothelial cell affinity.
Biomed Mater 3:44104
105. Kim TG, Park TG (2006) Biomimicking extracellular matrix: cell adhesive RGD peptide
modified electrospun poly(D, L-lactic-co-glycolic acid) nanofiber mesh. Tissue Eng 12:221
106. Park K, Ju YM, Son JS, Ahn KD, Han DK (2007) Surface modification of biodegradable
electrospun nanofiber scaffolds and their interaction with fibroblasts. J Biomater Science-
Polymer Edition 18:369
116 3 Results and Discussion

107. Yao C, Li XS, Neoh KG, Shi ZL, Kang ET (2008) Surface modification and antibacterial
activity of electrospun polyurethane fibrous membranes with quaternary ammonium
moieties. J Membr Sci 320:259
108. Ratner BD, Hoffman AS (2004) Nonfouling surfaces. In: Ratner BD, Hoffman AS, Schoen
FJ, Lemons JE (eds) Biomaterials Science: An Introduction to Materials in Medicine.
Elsevier Academic press, San Diego, pp 197–200
109. Patrucco E, Ouasti S, Vo CD, De Leonardis P, Pollicino A, Armes SP, Scandola M,
Tirelli N (2009) Surface-initiated ATRP modification of tissue culture substrates:
poly(glycerol monomethacrylate) as an antifouling surface. Biomacromolecules 10:3130
110. Patten TE, Matyjaszewski K (1998) Atom transfer radical polymerization and the synthesis
of polymeric materials. Adv Mater 10:901
111. Matyjaszewski K, Xia J (2001) Atom transfer radical polymerization. Chem Rev 101:2921
112. Xu FG, Neoh KG, Kang ET (2009) Bioactive surfaces and biomaterials via atom transfer
radical polymerization. Prog Polym Sci 34:719
113. Fu GD, Lei JY, Yao C, Li XS, Yao F (2008) Core-sheat nanofibers from combined atom
transfer radical polymerization and electrospinning. Macromolecules 41:6854
114. Sun X-Y, Shankar R, Borner HG, Ghosh TK, Spontak RJ (2007) Field-driven
biofunctionalization of polymer fiber surfaces during electrospinning. Adv Mater 19:87
115. Li D, Frey MW, Vynias D, Baeumner AJ (2007) Availability of biotin incorporated in
electrospun PLA fibers for streptavidin binding. Polymer 48:6340
116. Kim SJ, Lee CK, Kim SI (2005) Effect of ionic salts on the processing of poly(2acrylamido-
2-methyl-1-propane sulfortic acid) nanofibers. J Appl Polym Sci 96:1388
117. Haigh R, Rimmer S, Fullwood NJ (2000) Synthesis and properties of amphiphilic networks. 1:
the effect of hydration and polymer composition on the adhesion of immunoglobulin-G to
poly(laurylmethacrylate-stat-glycerolmonomethacrylate-stat-ethylene-glycol-dimethacrylate)
networks. Biomaterials 21:735
118. Mequanint K, Patel A, Bezuidenhout D (2006) Synthesis, swelling behavior and
biocompatibility of novel physically cross-linked polyurethane-block-poly(glycerol
methacrylate) hydrogels. Biomacromolecules 7:883
119. Burkersroda F, Schedl L, Gopferich A (2002) Why degradable polymers undergo surface
erosion or bulk erosion. Biomaterials 23:4221
120. Li S (1999) Hydrolytic degradation characteristics of aliphatic polyesters derived from
lactic and glycolic acid. J Biomed Mater Res 48:342
121. Ikada Y, Jamshidi K, Tsuji H, Hyon SH (1987) Stereocomplex formation between
enantiomeric poly(lactides). Macromolecules 20:904
122. Li S, Garreau H, Vert M (1990) Structure-property relationships in the case of the
degradation of massive aliphatic poly-(a-hydroxy acids) in aqueous media, part
2:degradation of lactide-glycolide copolymers: PLA37.5GA25 and PLA75GA25. J Mater
Sci Mater Med 1:131
123. Li S, Garreau H, Vert M (1990) Structure-property relationships in the case of the
degradation of massive aliphatic poly-(a-hydroxy acids) in aqueous media, part 3: influence
of the morphology of poly(L-lactic acid). J Mater Sci Mater Med 1:198
124. Li S, Garreau H, Vert M (1990) Structure-property relationships in the case of the
degradation of massive aliphatic poly-(a-hydroxy acids) in aqueous media, part 1: poly
(D, L-lactic acid). J Mater Sci Mater Med 1:123
125. Den Dunnen WFA, Robinson PH, van Wessel R, Pennings AJ, van Leeuwen M,
Schakenraad JM (1997) Long-term evaluation of degradation and foreign-body reaction of
subcutaneously implanted poly(DL-lactide-e-caprolactone). J Biomed Mater Res 36:337
126. Vert M, Li SM, Spenlehauer G, Guerin P (1992) Bioresorbability and biocompatibility of
aliphatic polyesters. J Mater Sci Mater Med 3:432
127. Li WJ, Cooper J, Mauck RL, Tuan RS (2006) Fabrication and characterization of
six electrospun poly(a-hydroxy ester)-based fibrous scaffolds for tissue engineering
applications. Acta Biomater 2:377
References 117

128. Zong X, Ran S, Kim K-S, Fang D, Hsiao BS, Chu B (2003) Structure and morphology
changes during in vitro degradation of electrospun poly(glycolide-co-lactide) nanofiber
membrane. Biomacromolecules 4:416
129. Xu CY, Inai R, Kotaki M, Ramakrishna S (2004) Aligned biodegradable nanofibrous
structure: a potential scaffold for blood vessel engineering. Biomaterials 25:877
130. Zhang XH, Baughman CB, Kaplan DL (2008) In vitro evaluation of electrospun silk fibroin
scaffolds for vascular cell growth. Biomaterials 29:2217
131. Li M, Guo Y, Wei Y, MacDiarmid AG, Lelkes PI (2006) Electrospinning polyaniline-
contained gelatin nanofibers for tissue engineering applications. Biomaterials 27:2705
132. Zhang J, Qi HX, Wang HJ, Hu P, Ou LL, Guo SH, Li J, Che YZ, Yu YT, Kong DL (2006)
Engineering of vascular grafts with genetically modified bone marrow mesenchymal stem
cells on poly (propylene carbonate) graft. Artif Organs 30:898
133. Chiu JB, Liu C, Hsiao BS, Chu B, Hadjiargyrou M (2007) Functionalization of poly(L-lactide)
nanofibrous scaffolds with bioactive collagen molecules. J Biomed Mater Res 83A:1117
134. Williamson MR, Black RA, Kielty C (2006) PCL-PU composite vascular scaffold
production for vascular tissue enginerring: attachment, proliferation and bioactivity of
human vascular endothelial cells. Biomaterials 27:3608
135. Zeng J, Xu X, Chen X, Liang Q, Bian X, Yang L, Jing X (2003) Biodegradable electrospun
fibers for drug delivery. J Controlled Release 92:227
136. Loebsack AB, Halberstadt CR, Holder WD, Culberson CR, Beiler RJ, Greene KG,
Roland WD, Burg KJL (1999) The development of an embedding technique for polylactide
sponges. J Biomed Mater Res 48:504
137. Brown DA, Chou YF, Beygui RE, Dunn JCY, Wu BM (2005) Gelatin-embedded cell-
polymer constructs for histological cryosectioning. J Biomed Mater Res Part B-Appl
Biomater 72B:79
138. Holy CE, Yakubovich R (2000) Processing cell-seeded polyester scaffolds for histology.
J Biomed Mater Res 50:276
139. Baker BM, Mauck RL (2007) The effect of nanofiber alignment on the maturation of
engineered meniscus constructs. Biomaterials 28:1967
140. Pham QP, Sharma U, Mikos AG (2006) Electrospun poly(e-caprolactone) microfiber and
multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of
cellular infiltration. Biomacromolecules 7:2796
141. Corey JM, Gertz CC, Wang B-S, Birrell LK, Johnson SL, Martin DC, Feldman EL (2008)
The design of electrospun PLLA nanofiber scaffolds compatible with serum-free growth of
primary motor and sensory neurons. Acta Biomater 4:863
142. Foroni L, Dirani G, Gualandi C, Focarete ML, Pasquinelli G (2010) Paraffin embedding
allows effective analysis of proliferation, survival and immunophenotyping of cells cultured
on poly(L-lactic acid) electrospun nanofiber scaffolds. Tissue Eng Part C Methods
16(4):751
143. Focarete ML, Gualandi C, Scandola M, Govoni M, Giordano ED, Foroni L, Valente S,
Pasquinelli G, Gao W, Gross RA (2010) Electrospun scaffolds of a polyhydroxyalkanoate
consisting of x-hydroxylpentadecanoate repeat units: fabrication and in vitro biocompatibility
studies. J Biomater Sci Polym Edition 21:1283
144. Van der Meulen I, de Geus M, Antheunis H, Deumens R, Joosten EAJ, Koning CE, Heise A
(2008) Polymers from Functional Macrolactones as Potential Biomaterials: Enzymatic Ring
Opening Polymerization, Biodegradation, Biocompatibility. Biomacromolecules 9:3404
145. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H (1984) Cell cycle analysis of
a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody
Ki-67. J Immunol 133:1710
146. Chen CS, Mrksich M, Huang S, Whitesides GM, Ingber DE (1997) Geometric control of
cell live and death. Science 276:1425
147. Huang S, Ingber DE (2000) Shape-dependent control of cell growth, differentiation and
apoptosis: switching between attractors in cell regulatory networks. Exp Cell Res 261:91
118 3 Results and Discussion

148. Griffith LG, Swartz MA (2006) Capturing complex 3D tissue physiology in vitro. Nat Rev
Mol Cell Biol 7:211
149. Hutmacher DW, Horch RE, Loessner D, Rizzi S, Sieh S, Reichert JC, Clements JA, Beler JP,
Arkudas A, Bleiziffer O, Kneser U (2009) Translating tissue engineering technology platforms
into cancer research. J Cell Mol Med 13:1417
150. Pampaloni F, Reynaud EG, Stelzer EHK (2007) The third dimension bridges the gap
between cell culture and live tissue. Nat Rev Mol Cell Biol 8:839
151. Yamada KM, Cukierman E (2007) Modelling tissue morphogenesis and cancer in 3D. Cell
130:601
152. Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diversity and escape
mechanisms. Nat Rev Cancer 3:362
153. Rockwood DN, Akins RE, Parrag IC, Woodhouse KA, Rabolt JF (2008) Culture on
electrospun polyurethane scaffolds decreases atrial natriuretic peptide expression by
cardiomyocytes in vitro. Biomaterials 29:4783
154. Schnell E, Klinkhammer K, Balzer S, Brook G, Klee D, Dalton P, Mey J (2007) Guidance
of glial cell migration and axonal growth on electrospun nanofibers of poly-e-caprolactone
and a collagen/poly-e-caprolactone blend. Biomaterials 28:3012
155. Xie J, Willerth SM, Li X, Macewan MR, Rader A, Sakiyama-Elbert SE, Xia Y (2009) The
differentiation of embryonic stem cells seeded on electrospun nanofibers into neural
lineages. Biomaterials 30:354
156. Lee CH, Shin HJ, Cho IH, Kang Y-M, Kim IA, Park K-D, Shin J-W (2005) Nanofiber
alignement and direction of mechanical strain affect the ECM production of human ACL
fibroblast. Biomaterials 26:1261
Chapter 4
Conclusions

Tissue engineering (TE) is a rapidly growing discipline which integrates the basic
principles of biology, engineering and material science with the aim to repair or
regenerate damaged tissues. To this scope, a cell-construct is engineered in vitro
by using a porous 3D material as cell culture support, commonly referred to as
scaffold. The role of the scaffold is to act as a temporary template, guiding cell
organization, growth and differentiation and providing a structural stability and a
3D environment where cells can produce new biological tissue. Therefore, the
scaffold must be designed to be bioresorbed in the organism and replaced by new
tissue produced by cells. The obtainment of a successful engineered tissue
encompasses the optimization of several critical elements (e.g. cell type, scaffold
material and 3D structure, cell culture conditions, etc.) and an effective multi-
disciplinary approach involving not only biological and medical expertises but also
competences in engineering, chemistry and materials science is imperative in this
context.
The present Ph.D. project focused its attention on the design, fabrication,
manipulation and characterization of innovative polymeric bioresorbable scaffolds
made of hydrolysable polyesters. Two techniques were employed to fabricate
scaffolds: supercritical carbon dioxide (scCO2) foaming and electrospinning (ES).
Microporous 3D foamed materials, that can assume a structural supporting role
when used as tissue replacements, will be particularly suitable in hard TE
(e.g. bone or cartilage TE), whereas flexible nanofibrous electrospun mats will be
more appropriate for replacement of soft tissues such as cardiac, vascular or
nervous tissues.
ScCO2 foaming is a powerful scaffold fabrication technique since it does not
require the use of solvents that can be toxic to mammalian cells. However,
applicability of this technology is mainly limited to amorphous polymers which
can be easily processed into foams. In the course of the present Ph.D., it was
demonstrated that a proper modification of the scCO2 foaming process usually
applied to amorphous materials enables to successfully produce porous structures

C. Gualandi, Porous Polymeric Bioresorbable Scaffolds for Tissue Engineering, 119

Springer Theses, DOI: 10.1007/978-3-642-19272-2_4,
Ó Springer-Verlag Berlin Heidelberg 2011
120 4 Conclusions

also from highly crystalline polymers. The investigation of the effect of process
parameters on scaffold morphology by Micro X-ray Computed Tomography
contributed to demonstrate the versatility of this technique and to broaden the
range of polymeric materials that can be processed into foams using scCO2 as
porogen (Sect. 3.1).
The present research activity mainly focused onto the study and the develop-
ment of ES process and of electrospun products. The main benefit in using ES
technique is its outstanding versatility: it is documented that electrospun fibres
from over two hundred synthetic and natural polymers can be produced. Another
advantage is the simplicity of the instrumental apparatus that does not require any
sophisticated and expensive equipment. Moreover, given the intrinsic morpho-
logically biomimetic features of electrospun materials, which are composed of
sub-micrometric fibres resembling the fibrous component of native tissue, they are
considered the most promising scaffolds by most of the scientific community. In
the course of the present Ph.D., a highly reproducible ES process, performed in a
glove box specifically designed to control the environmental conditions, was
developed for fabricating innovative electrospun scaffolds. New non-commercial
polymers were electrospun for the first time. These polymers, being more
hydrophobic than common commercial bioresorbable polyesters, might be par-
ticularly interesting for long-term applications. Innovative patterned electrospun
non-woven meshes were also produced by using novel ad hoc designed ES col-
lectors that enabled a fine control of fibre deposition geometry. Particularly
interesting is the possibility to obtain different fibre patterns contemporarily
available in a given scaffold. Such type of scaffold is suitable for further inves-
tigation concerning the effect of substrate morphology on cell homing, spreading
and organization.
Besides the improvement of ES process, part of the research activity concerned
the production of bio-functionalized electrospun scaffolds that was achieved by
either a bulk functionalization or a surface functionalization. A bulk functionali-
zation was successfully performed by incorporating a growth factor (GF) within
electrospun fibres demonstrating that ES technology is suitable for producing
bioactive nanofibrous scaffolds containing GFs, which can be subsequently
released without loss of bioactivity. The surface modification approach developed
in the course of the present research activity aimed at controlling cell adhesion on
electrospun scaffolds. The modification was achieved by covering fibre surface
with highly hydrophilic polymer brushes carrying hydroxyl groups. The hydro-
philic nature of the brushes is expected to passivate fibre surface towards protein
absorption, and thus towards cell attachment. The preliminary results presented in
this thesis open a wide range of possibility to exploit the hydroxyl groups of the
brushes for functionalizing fibre surface with biomolecules, such as peptides or
GFs, for eliciting the desired cell behaviour.
Collaboration with biochemical laboratories allowed to perform cell culture
experiments by using some of the electrospun scaffolds produced in the course of
this Ph.D. Finally, the last part of the research activity was dedicated to explore the
possibility to use electrospun materials in an application alternative to TE that will
4 Conclusions 121

probably have a great impact in the future of life sciences. Indeed, very recently,
3D scaffolds have been employed for the development of new in vitro tumour
models. Preliminary cell culture experiments carried out in the course of this
research demonstrated a strong influence of fibre orientation on cancer cell col-
lective behaviour. This finding might provide a key information for better
understanding the role of natural ECM on tumour malignity in vivo. However,
results provided in the present thesis are far from being exhaustive and cancer cell
behaviour on 3D electrospun scaffolds is worth of further thorough investigation.